CN101657097A - With the inflammation is the treatment of diseases of feature - Google Patents

Abstract

The present invention partly provides method, nucleic acid, carrier, albumen and the binding molecule that can be used to regulate as approach such as complement pathways.In addition, these method and compositions can also be used for the research and/or the treatment of the various patient's condition or the disease relevant with complement pathway.

Description

With the inflammation is the treatment of diseases of feature

Related application

The application requires No. the 60/892nd, 395, U.S. Provisional Application submitting on March 1st, 2007 and the rights and interests of No. the 60/985th, 024, the U.S. Provisional Application submitted on November 2nd, 2007, and this paper is by with reference to introducing its whole disclosures.

Background technology

Complement system is the important component part (Volanakis summary, 1998) of natural immune system and adaptive immune system.Complement plays an important role in microorganism killing, and for the transhipment of immune complex with remove most important.Many activation products of complement system are also relevant with short scorching function or immunoloregulation function.Complement system is made up of the relevant albumen that waits with film of blood plasma, and described albumen constitutes three kinds of enzymes and activates cascade: classical pathway, lectin pathway and alternative route.All three kinds of approach can both cause forming terminal compleoment complex (TCC) and a collection of bioactive product.

In some cases, complement activation is by identification and in conjunction with the specific antibodies of multiple pathogene and foreign molecules and/or by the direct interaction startup of complement protein and foreign substance.When activating, these approach cause forming labile protein multienzyme complex C3-invertase.Classical pathway C3-invertase (C4b2a) and alternative route C3-invertase (C3bBb) thus the α chain that can both cut C3 generates C3b.C3b has and the covalently bound potential of biological surface.C3b is in conjunction with the phagocytotic conditioning that causes polymorphonuclear cell and macrophage.When extra C3b can utilize, the C3-invertase can be brought into play the function of C5-invertase, cutting C5 also starts TCC or the assembling of membrane attack complex (MAC), and described membrane attack complex comes the mediated cell cracking by the pore-forming protein compound is inserted in the targeted cells film.

The precise function of complement system depends on tight adjusting, because the activation of complement cascade can cause many generations of facilitating the albumen of inflammation.This is favourable when helping host defense, if but be activated in autologous tissue then be harmful to.Usually, the activation of C3 is maintained at low-levelly in the blood, and the deposition of C3b is limited in the surface of pathogene.

In order to regulate complement system, many Complement Regulatory Protein play the function of restriction complement activation.These albumen and C3 or C4 derivative interact and by the closely linked gene code (Diaz-Guillen etc., 1999) that comprises complement activation instrumentality (RCA) gene cluster on the human chromosome 1q32.

Complement factor H (CFH or fH) is the plasma protein by a kind of RCA gene code, and is solubility activation inhibitor (Muller-Eberhard etc., 1980 that substitute complement pathway; Zipfel etc., 2002; Rodriguez de Cordoba etc., 2004).CFH can prevent combining of factor B and C3b, demonstrate and quicken active the decay of dissociating of C3bBb compound, serve as the co-factor to the cutting of C3b by factor I, and generation (Whaley and Ruddy, 1976 of blocking-up C5b6-9 (being also referred to as membrane attack complex (MAC)); Weiler etc., 1976; Pangburn etc., 1977).CFH can and interact with the multiple part combination that comprises C3b, heparin, bacterium surface albumen, acute phase protein, C-reactive protein (CRP), adrenomedulin and cell surface receptor (Zipfel etc., 2002).

A member of the protein family that people CFH is made up of seven kinds of structures and the multi-functional haemocyanin of immunologically-mediated multiple domain.These albumen comprise CFH and factor H sample albumen 1 (FHL-1) and five kinds of factor H associated protein (FHR-1 to FHR-5).These albumen ad hoc are made up of short consensus repeat (SCR) or complement control module separately, each exons coding that freely separates of these short consensus repeats and complement control module.CHF (150kDa) is made up of 20 SCR.The FHL-1 (43kDa) that forms by 7 SCR territories by alternative splicing from CFH derive (Estaller etc., 1991; Sim etc., 1993).Similar to CFH, FHL-1 exercises the function of complement instrumentality and demonstrates co-factor and decay acceleration active (Zipfel and Sherka, 1999).In addition, because the RGD territory that exist to expose in SCR4, so FHL-1 has and comprises the unique function (Hellwage etc., 1997) of serving as attachment proteins.CFH is present in people's the blood plasma with 500 μ g/ml.By contrast, the plasma concentration of FHL-1 is 10 μ g/ml～50 μ g/ml.Five kinds of FHR albumen is derived from the independent gene in the RCA gene cluster separately.Although the member of this family is different on the number of SCR, individual SCR territory demonstrates high homology each other.

The albumen territory that CFH and associated protein are assembled into a plurality of each autofolding has shown a kind of structure/functional relationship.The complement of CFH and FHL-1 is regulated the territory and is positioned at four amino terminal SCR.Three C3 are arranged in CFH in conjunction with the territory.The terminal territory of N (SCR 1～4) combines (Gordon etc., 1995 with complete C3b; Kuhn etc., 1995; 1996), intermediate field (SCR 12～14) combines with the C3c fragment, and the terminal territory of C (SCR19～20) combines (Jokiranta etc., 2000) with C3d.The cracking function (Zipfel etc., 2002) of C5b6-9 TCC has also been blocked in the terminal territory of C (SCR19～20).FHL-1 does not have this function.CFH and FHL-1 contain the overlapping binding site to heparin, C-reactive protein (CRP) and M-albumen (Giannakis etc., 2003) that is arranged in SCR7.

Albumen in this family is mainly synthetic in liver, but CFH also expresses (Friese etc., 1999) in as multiple different cell types such as peripheral blood lymphocyte, sarcoblast, fibroblast, neuron and Deiter's cells according to the show.Recently, in being derived from human retina PE (RPE) cell and choroidal expressed sequence tag storehouse, CFH sequence (Wistow etc. have been identified, 2002), and immunohistochemical staining in the choroid pipe He in the RPE fringe region, identified CFH (Klein etc., 2005).

The CFH polymorphism increases relevant (Edwards etc., 2005 with the risk of senile macular degeneration (AMD); Klein etc., 2005; Haines etc., 2005).This polymorphism, promptly the tyrosine at 402 amino acids places to the variation (tyr402his or Y402H) of histidine but be the reason of the attribution risk of about 50% AMD.The Y that identifies in the recent Science article → H polymorphic position (Edwards etc., 2005 in SCR7; Klein etc., 2005; Haines etc., 2005).

Known CRP can activate CCP, and by directly suppress the deposition (Mold etc., 1999) of C5b6-9 in conjunction with CFH.By substitute the neutral tyrosine CFH (Rodriguez de Cordoba etc., 2004) that combines with heparin and/or CRP of change potentially with positively charged histidine.In clinical research, in AMD patient, observed the change of serum C RP level (Seddon etc., 2004) that raises compared with the control.In addition, slowed down the progress of AMD with the nutritional supplementation of zinc and biochemical research shows that the CFH function is to zinc concentration sensitivity (AREDS seminar, 2001; Blom etc., 2003).Therefore, replace level of inflammation (Edwards etc., 2005 that CFH and the change that combines of CRP or heparin on the retinal surface cause may influence outer retina by tyr402his; Klein etc., 2005; Haines etc., 2005).

The description that in the human and animal, all has couple CFH to lack.They are by causing damaging the brachymemma of CFH function or the sudden change of aminoacid replacement causes (Ault etc., 1997; Sanchez-Corral etc., 2002; Hegasy etc., 2003).The shortage of CFH has caused the not controlled activation of alternative complement pathway and consumption (the Thompson ﹠amp of C3 and other terminal complement component in the blood plasma; Winterborn, 1981; Ault etc., 1997).People's dysfunction CFH molecule is relevant with two kinds of different ephrosis: membrano proliferative glomerulonephritis (MPGN) and atypia hemolytic uremic syndrome (Wyatt etc., 1982; Ault, 2000; Zipfel, 2001).In the patient's who suffers from II type MPGN eye, found with AMD in the identical drusen of composition found.The manhood appearance in early days when ephrosis occurs usually of this drusen is than obviously more Zao (Mullins etc., 2001 in AMD; Colville etc., 2003).In addition, the animal of CFH shortage is developed ephrosis (Hogasen etc., 1995 that to have the MPGN feature; Pickering etc., 2002).In pig, CFH lacks and to have caused similar the carrying out property glomerulonephritis (Hogasen etc., 1995) with the people II type MPGN that can cause kidney failure.Similarly, the mouse that CFH knocks out is spontaneously developed the glomerulonephritis (Pickering etc., 2002) that to be similar to equally people II type MPGN.In possible MODEL C cl-2-defective (chemokine ligand 2) mouse of AMD or Ccr-2-defective (Ccl-2 acceptor) mouse, detect the drusen of forming by complement and immunoglobulin deposit thing, thereby showing that rodent can be developed drusen (Ambati etc., 2003).

Complement activation is relevant with several different human diseasess that comprise atherosclerotic and Alzheimer's disease.The composition vitronectin that enriches in the drusen also is and atherosclerotic (Niculescu etc., 1989), amyloidosis (Dahlback etc., 1993), the composition of born of the same parents' external sediment thing that elastosis (Dahlback etc., 1988) and II type MPGN (Jansen etc., 1993) are relevant.Vitronectin is the multifunctional protein (Preissner, 1991) that works in the lysis that cell adhesion, hemostasis are safeguarded and the inhibition complement is induced.In addition, atherosclerotic plaque is also total as various other components such as complement component and apo Es with drusen.In epidemiological study, reported late period AMD with carotid atherosclerosis between related (Vingerling etc., 1995), and another has determined significant correlation (Blumenkranz etc., 1986) between AMD patient's the elastic tissue degeneration of the corium that does not expose in the sun and the CNV.At last, in drusen, also found amyloid beta peptide, the main component of the neuritic plaques in the Alzheimer's disease (Johnson etc., 2002).Amyloid beta peptide is prompted as main complement activation agent (Bradt etc., 1998).

Complement system has mediated these inflammation performances, and many stimulations can trigger the activation of complement system.For example, the TGF beta molecule is induced the expression of some complement factor and is suppressed the expression of other complement factor simultaneously.

The common cause that senile macular degeneration (AMD) is that the over-65s individual visual descends in the developed country.The feature of dryness AMD is to cause the carrying out property sex change of the macula lutea of central field of vision visual loss.About 25% 65 years old～74 years old individuality has dryness AMD to a certain degree, and increases to 40% (Hamdi ﹠amp at 75 years old～84 years old incidence of disease; Kenney, 2003).In the U.S., estimate at 1,000 ten thousand people and descend, and along with the increase at crowd's age because of AMD suffers from eyesight, be in (Hamdi ﹠amp in the risk 2,100 ten thousand people of the U.S.; Kenney, 2003).More acute weak property AMD is called moist AMD, comprises in the retina red angiogenesis and exosmoses.At present AMD there is not effective therapy.

Similar to many other chronic diseases, AMD is caused by the combination of genetic risk factor and environmental risk factor.These risk factors comprise age, smoking and family history (AREDS seminar, 2000).But inherent cause shows as the autosomal dominant proterties in most affected individuals.

The feature of AMD is (Pauleikhoff etc., 1990 gathered that are positioned at drusen between retinal pigment epithelium (RPE) basilar memebrane and Bruch's membrane (Bruch ' s membrane) internal layer; Bressler etc., 1990).Other age that drusen and contiguous Bruch's membrane occur, relevant variation was facilitated RPE and amphiblestroid functional disorder and sex change (Bird summary, 1992) by causing nutrition and waste exchange between ischemic and restriction retina and the choroid.The developing immune-mediated process of AMD has been pointed out in several researchs.Importantly, predict, in AMD patient's serum, detected autoantibody (Penfold etc., 1990) as the hypothesis that development and/or elimination to drusen relate to immune and inflammation mediated process.

To people's drusen and in the drusen side or press the molecular analysis-by-synthesis of RPE cell thereon to demonstrate immunoreactivity (Johnson etc., 2000) to immunoglobulin and the complement system composition relevant with immune complex deposit.Drusen also contain in immune system, work as vitronectin (Hagemen etc., 1999) and apo E multifunctional proteins such as (Anderson etc., 2001).In addition, in drusen, also identify the molecule (Mullins etc. that the acute stage of inflammation replied as participations such as amyloid P composition and alpha1-antitrypsins, 2000), and the albumen (factor X, fibrin ferment and fibrinogen) (Mullins etc., 2000) that participates in blood coagulation and fibrinolytic.Help the chronic inflammation that complement cascade activates is replied (Hageman etc., 2001 according to showing that drusen forms with relevant RPE pathology; Johnson etc., 2001).

Cause and cause the another kind of retina disturbance to look obstacle to be ground pattern atrophy (Geographic Atrophy) by AMD, it can cause the death of (patch) in blocks of rod cell and cone cell and RPE cell.

Should be with not quoting or discussing and be considered as approving that it is a prior art of the present invention reference herein.

Summary of the invention

The present invention partly provides the method and composition that is used to regulate the immunology approach.The present invention partly relates to molecule is transported to cell.Described cell can be in external or body.Some embodiment of the present invention relates to the adjusting complement pathway, for example, and classical pathway, lectin pathway or alternative route.

The present invention partly provides the method for adjusting (for example, suppressing) complement pathway and/or complement relevant disease.In addition, the inventor has described composition and the method that is used to regulate complement pathway in this article.To the adjusting of complement pathway can be used for regulating the inherent mechanism of morbid state, the study of disease of animal, as the control group (control arm) of the research relevant and be used at complement pathway generation heterogeneity and/or end product with complement.Some embodiment of the present invention can be used for studying immunology approach, research relevant disease state, exploitation to the treatment of morbid state, animal or at external establishment morbid state (for example, development model in as animals such as mouse or rats) or be used for screening of medicaments.Some embodiment of the present invention relates to the adjusting to CCP, alternative complement pathway or agglutinin complement pathway.

This paper provides various compositions and/or the method that is used to regulate as immunology approach such as complement pathways.

In addition, the inventor provides the composition/molecule that is used to suppress complement pathway in this article.For example, the inventor has been found that some analog of factor B weakens complement activation by the compound that keeps C3bB and factor D.So this paper provides the example that demonstrates the factor B analog that can suppress complement activity.The present invention also provides the factor D analog that can weaken complement activation similarly.These analogs can provide and weaken but thoroughly do not block the advantage of complement activation.

In some embodiments, molecule is carried to determine its for example effect on the part of eye or shortage.In some embodiments, molecule is carried so that beneficial effect or the result of treatment to the people for example to be provided.In some embodiments, molecule of the present invention (for example albumen) is used for studying biological approach or morbid state so that screening of medicaments or as the contrast of research and/or test.In some embodiments, complement pathway can be regulated, strengthens, mediates or suppressed to molecule, for example, and classical pathway, lectin pathway or alternative route.In some embodiments, molecule of the present invention is peptide, albumen, complement inhibitor or nucleic acid.

In addition, the invention provides the whole bag of tricks that is used for molecule of the present invention for example is transported to the specific part/zone of eye or eye.Method of the present invention has also been considered for example as described herein molecule to be carried out one or many transmission.

Some embodiment of the present invention provides and has been used for studying, has suppressed, stablizes, increases the weight of (for example, producing disease model in animal), healing, treatment, prevent disease or the patient's condition; Weaken the seriousness of the disease or the patient's condition; Shorten the course of disease of the disease or the patient's condition; Improve or change the method and/or the composition of pathology, symptom or the symptom of the disease or the patient's condition.In some embodiments, the disease or the patient's condition that the disease or the patient's condition are complement-mediated, complement is relevant, complement is relevant or complement relies on.In some embodiments, the disease or the patient's condition be the disease that causes of blind property illness in eye, inflammation, early-stage senile macular degeneration, senile macular degeneration (AMD), moist AMD, glaucoma, uveitis, pattern atrophy, diabetic proliferative retinopathy become and other disease as herein described or the patient's condition.

The present invention also provides the method and composition that is used to block, suppress, strengthen and/or regulate following situation: (i) relate to the reaction of C3b and factor B; (ii) relate to the reaction of C3bB and factor D; (iii) relate to the reaction of C3b, factor B and factor D; The (iv) cutting of factor B (for example, passing through factor D); (the v) cutting of C3 (for example, passing through factor B); (vi) factor D from C3bBD dissociate and/or (vii) complement pathway for example substitutes complement pathway.

Some embodiment of the present invention provides for example synthesizing, cutting or the active method for the treatment of, improving or prevent the disease of factor B mediation in the study subject by inhibiting factor B.

The present invention also provides the mutant that comprises complement pathway composition (as factor B) for example as described herein or the albumen of variant.Some embodiment of the present invention provides the variant of the factor B with one or more following features: the ability of the cutting C3 of reduction, the ability of being cut by factor D that combines more closely with factor D, combines more closely or reduce with C3b.

The present invention includes the molecule that (i) combines with factor C3b and factor D simultaneously, for example, bispecific antibody fB3 etc.; The (ii) complement protein analog that increases with the combination (comparing) of factor C3b and factor D simultaneously with its native form; The (iii) complement protein analog that the combination (comparing with its native form) of factor D is increased; The (iv) complement protein analog that the combination (comparing with its native form) of C3bB compound is increased.The present invention also comprises the method for using molecule of the present invention (as the molecule of the described i～iv of this section) to suppress complement pathway.

Some embodiment of the present invention provides the method for the disease of treatment complement-mediated, and described method comprises that the patient to the needs treatment uses and comprises the molecule that suppresses complement activity and/or comprise the genetically modified carrier of the described molecule of encoding and/or comprise the pharmaceutical composition of the cell of the carrier of expressing described molecule.In some embodiments, described molecule is the complement factor analog.

In some embodiments, complement factor comprises the function of one or more changes.In some embodiments, the complement factor of change comprises the proteinase activity that weakens.In some embodiments, described complement factor is a factor B.In some embodiments, the complement factor B analog comprises the C3b binding affinity of comparing increase with unaltered complement factor.This realizes in conjunction with the change in the territory by the C3b such as replacement as aspartic acid and/or asparagine in some cases.In some embodiments, aspartic acid is substituted by glycine, alanine or asparagine.In some embodiments, asparagine is substituted by glycine, alanine or aspartic acid.In some embodiments, this aspartic acid corresponding to 279 amino acids of SEQ ID NO:2 this asparagine corresponding to 285 amino acids of SEQ ID NO:2.

In some embodiments, factor B comprises the interior change of avtive spot of serine protease domain.In some embodiments, serine protease domain comprises corresponding to 739～746 the amino acid of SEQ ID NO:2 or by 739～746 amino acid corresponding to SEQ ID NO:2 and forms.In some cases, change and for example to comprise and replace aspartic acid with serine, tyrosine, glycine, alanine, glutamic acid or asparagine.In some embodiments, substituted aspartic acid is corresponding to 740 amino acids of SEQ ID NO:2.In some embodiments, the complement factor B analog comprises SEQ ID NO:4 or comprises 26～764 amino acids of SEQ ID NO:4.

In some embodiments, the complement factor B analog has and compares the ability of being cut by factor D that weakens with natural factor B.In some embodiments, the factor B analog is included in the change in the factor D cleavage site.In some embodiments, described change for example comprises with alanine at least one lysine and/or arginic each replacement.In some embodiments, described at least one lysine is corresponding to 258 of SEQ ID NO:2 or 260 amino acids and described arginine 259 amino acids corresponding to SEQ ID NO:2.

The present invention also provides to have and has compared proteolytic activity that weakens and/or the C3bBb binding affinity of comparing increase with natural Complement Factor D with natural Complement Factor D.In some embodiments, the Complement Factor D analog comprises the ability of comparing the cutting factor B of reduction with natural Complement Factor D.In some embodiments, the Complement Factor D analog comprises the change in the serine protease catalytic domain of fD.In some embodiments, the change in the serine protease catalytic domain of fD comprises: (i) corresponding to His66, the Asp114 of SEQ ID NO:27 (people fD) or amino acid whose replacement or the disappearance of Ser208; Or (ii) be close at least one amino acid whose insertion of His66, Asp114 or the Ser208 of SEQ ID NO:27.In some embodiments, replace amino acid with at least one neutral amino acid, amino acid that at least one is electronegative or at least one nonpolar amino acid corresponding to His66.In some embodiments, replace amino acid with at least one positively charged amino acid or at least one nonpolar amino acid corresponding to Asp114.In some embodiments, replace amino acid with at least one charged amino acid or at least one nonpolar amino acid corresponding to Ser208.In some embodiments, the Complement Factor D analog is compared at the N end with wild type factor D and is comprised one or more extra amino acid.In some embodiments, described one or more additional amino acid comprises glycine and arginine.

Some embodiment of the present invention provides the method for the disease of treatment complement-mediated, and described method comprises that the patient to the needs treatment uses and comprises: the Complement Factor D analog that (i) suppresses or reduce complement activity; (ii) the encode carrier of described Complement Factor D analog; Or (iii) contain the pharmaceutical composition of cell of the described carrier of the described Complement Factor D analog of encoding.

In some embodiments, the molecule of inhibition complement activity is the molecule of the conjugated complement factor.In some embodiments, this molecule comprises at least one complementary determining region of the antibody of the conjugated complement factor.In some embodiments, described molecule is antibody or its fragment of the conjugated complement factor.In some embodiments, antibody is human antibodies, humanized antibody, chimeric antibody, rodent antibody, chicken antibody or rabbit antibody.In some embodiments, binding molecule is fit (aptamer) of the conjugated complement factor.In some embodiments, but binding molecule binding factor B of the present invention, factor C3b or factor D.

In some embodiments, the disease of complement-mediated is an eye disease.In some embodiments, with pharmaceutical composition of the present invention for example via injection, cornea local injection in intravitreal injection, subretinal injection, the camera oculi anterior or use, injection (subtenoninjection) or eye drops come delivered/administered to arrive eye down for subconjunctival injection, capsula bulbi.

The present invention also provides the factor B analog of the C3b binding affinity that comprises the proteinase activity that weakens and change.Some embodiment of the present invention provides the Complement Factor D that comprises the proteinase activity that weakens analog.

Some embodiment of the present invention provides the method for the treatment of mammiferous disease, and described method comprises the pharmaceutical composition that administration is comprised the molecule of inhibition or reduction complement activity.In some cases, described molecule is albumen or nucleic acid.In some embodiments, described pharmaceutical composition comprises the carrier of the described molecule of encoding.In some embodiments, described albumen is the analog of complement pathway composition, for example, and factor D analog or factor B analog.In some embodiments, analog is people'sfactor B 1, B2 or B3 ormouse factor B 1, B2 or B3.

Some method of the present invention relates to treats or prevents the disease of complement-mediated or the method for illness, for example, wherein said disease is that drusen forms, macular degeneration, AMD, atherosclerotic, BDR, vitreoretinopathy, keratitis, airway hyperreactivity, with immune diseases associated, with the autoimmunity diseases associated, lupus nephritis, systemic loupus erythematosus (SLE), arthritis, rheumatism, antiphospholipid antibody syndrome, small intestine and kidney I/R damage, asthma, the atypia hemolytic uremic syndrome, II type membrano proliferative glomerulonephritis, the non-proliferative glomerulonephritis, pregnancy loss, glaucoma, uveitis, high intraocular pressure, brain damage, palsy, organ damage after the wound, organ damage after the infarct, vasculitis, ischemic damage and reperfusion damage, heart and lung Coronary Artery Bypass wound (for example, used in the open heart operations), cerebrovas-cularaccident, Alzheimer's disease, graft rejection, infect, septicemia, infectious shock;

Syndrome, myasthenia gravis, antibody-mediated skin disease, I type and type ii diabetes, thyroiditis, ITP and hemolytic anemia, neuropathy, multiple sclerosis, the cardiopulmonary bypass damage, polyarteritis nodosa, anaphylactoid purpura (Henoch Schonlein purpura), serum sickness, Goodpasture (Goodpasture ' s disease), systemic necrotizing vasculitis, post-streptococcal glomerulonephritis, idiopathic pulmonary fibrosis, membranous glomerulonephritis, myocardial infarction, acute shock lung syndrome, adult Respiratory Distress Syndrome(RDS), perfusion again, the disease of repulsion and/or complement-mediated.

Some method of the present invention comprises that the MCP with factor H,factor H sample 1, MCP, DAF, CD59 or soluble form uses separately, perhaps before complement factor analog of the present invention, use simultaneously afterwards or with it.

In some embodiments, adopt catalytic antibody to suppress complement activity.In some embodiments, catalytic antibody for example plays the effect of protease by complement proteins such as cutting factor B, factor D, factor B b, factor C3 and/or factor C3b.

In some embodiments, adopted and suppressed the RNA that complement protein is expressed, for example wherein RNA is ribozyme, antisense oligonucleotides, siRNA, miRNA or RNAi, and for example wherein said complement protein is C3, fB, fD, C5, C6, C7, C8 or C9.In some embodiments, pharmaceutical composition comprises the RNA of the expression of at least two kinds of different complement proteins of inhibition.

Some embodiment of the present invention provide can combine with factor C3b and factor D through isolated molecule, wherein said molecule is not natural factor B, fB1, fB2 or fB3.Some embodiment of the present invention provides the complement protein analog of comparing with its native form with factor C3b and factor D that increases that combines, and wherein said complement protein analog is not fB1, fB2 or fB3.Some embodiment of the present invention provides the complement protein analog of comparing with its native form with factor D that increases that combines, and wherein said complement protein analog is not fB1, fB2 or fB3.Some embodiment of the present invention provides the complement protein analog of comparing with its native form with the C3bB compound that increases that combines, and wherein said complement protein analog is not fB1, fB2 or fB3.In some embodiments, the complement protein analog has the combination of for example passing through at least 2 times increase of immune precipitation determination.

In some embodiments, carrier is retroviral vector, slow virus carrier, adenovirus vector, AAV carrier, herpesvirus vector, hepatitis viruse carrier (as hepatitis B carrier or fourth liver carrier), SV40 carrier or EBV carrier.In some embodiments, slow virus is HIV, EIAV, SIV, FIV or BIV.In some embodiments, viral vectors comprises decay accelerating factor.The present invention also provides the cell that produces viral vectors of the present invention.

In some embodiments of the present invention, can be before using pharmaceutical composition of the present invention, simultaneously and/or use antiinflammatory agent afterwards.In some embodiments, antiinflammatory agent is used in solution identical with described pharmaceutical composition place and/or identical syringe.In some embodiments, antiinflammatory agent is administered to eye.Antiinflammatory agent includes but not limited to sodium sulfo benzoate between dexamethasone, dexamethasone, dexamethasone sodium phosphate, rapamycin (rapamycin), FK506, fluorometholone (fluorometholone), bromfenac (bromfenac), pranoprofen (pranoprofen), RESTASIS^TM, cyclosporin ophthalmic emulsion, naproxen (naproxen), glucocorticoid, ketorolac (ketorolac), brufen, Tolmetin (tolmetin), NSAID (non-steroidal anti-inflammatory drug), steroidal anti-inflammatory medicine, Diclofenac (diclofenac), Flurbiprofen (flurbiprofen), Indomethacin (indomethacin) and suprofen (suprofen).

In some embodiments, suppress complement activity by first molecule that administration is suppressed complement activity and the carrier that coding suppresses second molecule of complement activity.In some cases, described first molecule is different with second molecule.In some cases, before using described carrier, simultaneously and/or use described first molecule afterwards.In some cases, described first molecule is used in same solution and/or identical syringe with described carrier.Sometimes described first molecule and/or described vector administration can be arrived eye.

Some embodiment of the present invention provides and has comprised the Complement Factor D of comparing the proteolytic activity that weakens with natural Complement Factor D.

In some embodiments of the present invention, with the carrier of the molecule of the present invention or the described molecule of encoding with weekly, every month, per 2 months, per 3 months, per 6 months, per 9 months, annual, approximately or at least once individuality was used in per 18 months, per 2 years, per 30 months, per 3 years, per 5 years or per 10 years, for example be administered to eye.In some embodiments, the carrier of the molecule of the present invention or the described molecule of encoding is applied once and is suppressing complement activity in the lifelong prolongation period more than one day, more than January or until described individuality.In other embodiments, with the carrier of the molecule of the present invention or the described molecule of encoding with weekly, every month, per 2 months, per 3 months, per 6 months, per 9 months, annual, be no more than once in per 18 months, per 2 years, per 30 months, per 3 years, per 5 years or per 10 years individuality used, for example be administered to eye.

Some embodiment of the present invention provides and (has for example carried coding molecule of the present invention, the treatment molecule) the vector construction body or the viral vectors of nucleic acid, coded molecule of the present invention complement inhibitor such as decay accelerating factor (DAF) are for example arranged or compare with wild type molecule lack or have the complement factor B analog of less biological function or specifically with participate in the binding molecule that complement function, approach or active molecule combine (for example fit, antibody, antibody sample molecule or antibody derived molecules) etc.In some embodiments, utilize the conversion visitor of vector construction body of the present invention or viral vectors that lasting conveying and/or expression from the molecule of cell in the retina for example are provided.

The present invention provides and has been used for the method for (in vivo or external) transformant, particular cell types or cell mass in addition.Some method of the present invention can be used for cell transformed and include but not limited to retina cell and/or RPE cell.Some method of the present invention can be used to transform sclera cell, keratocyte, iris cells, ciliary body cell, choroid cell, conjunctival cells, fascial bursa (tenonscapsule) cell, retina cell, retina undertissue cell, eye external fat cell, muscle cell (for example, extraocular muscle meat cell) and/or fascia tissue cell.Yet, the invention is not restricted to transform certain cell type.In some embodiments, described cell is in the animal interior certain organs or a separate space, as in brain, eye, spinal cord, joint, the circulatory system and/or in the blood.In some embodiments, molecule being transported to cell also is introduced into this cell in the animal subsequently.

Some embodiment of the present invention provides the method that is used to detect usefulness (efficacy) or measures usefulness, and described method comprises for example detection and/or mensuration complement activity and/or complement pathway composition and/or its activity after using composition of the present invention.In some embodiments, this can be continuously or periodically measures so that monitor disease states and/or be used for determining the process of further methods of treatment (relevant with methods described herein or other method).

What it is contemplated that is can take any other method as herein described or composition into account and implement any method as herein described or composition.In claims and/or specification, unite when using when " comprising " with term, word " one " or " a kind of's " use can refer to " one (kind) ", but also can be consistent with the implication of " (kind) or a plurality of (kinds) ", " at least one (kind) " and " (kind) or more than (kind) ".When with tabulation when together using, term/phrase " and/or " use be meant utilizable one or more listed content, for example, be not limited to one or all elements.

In following embodiment, will extra feature and advantage be described.

Description of drawings

For the purpose that the present invention will be described, be described in the drawings some embodiment of the present invention.Yet, the definite arrangement and the means of the embodiment that the invention is not restricted to describe in the accompanying drawing.

Fig. 1 has shown the example of describing the AMD development.The feature of early stage AMD is the deposition of drusen under retinal pigment epithelium (RPE) cell tier.Shallow point or white point during drusen is shown as among the figure.

Fig. 2 has shown and has been used for the example of preparation based on four constructs of the carrier of BIV (BIV).RSV is Rous sarcoma virus (RSV) promotor.CMV is cytomegalovirus (CMV) enhancer arranged side by side with the Zone R of 5 ' long terminal repeat (LTR).Thereby SIN indication from the enhancer in the U3 district of 3 ' LTR and the generation of the disappearance in the promotor from the inactivation carrier.

Be to instruct vector rna to be packaged into the interior packaging signal of virion.Heterologous gene (Heterologous Gene) is meant the expression cassette of the code area that comprises promotor and coding molecule (for example, treatment molecule).Gp64 is a baculoviral gp64 env gene.

Fig. 3 has shown that the green fluorescent protein (GFP) in the rat retina of biv vector transduction expresses.With among the 3μ l 3 * 10⁵Transduced unit (tu) is used through subretinal injection.After one month, put to death described rat and collect retina, and in retina, cut slit to prepare smooth complete sample.Fig. 3 A: the grey outline line has been described the retina edge.Injection site place and transduction on every side and GFP expression have been shown than bright area.Fig. 3 B has shown that the immunohistochemical staining of GFP is mainly in the RPE layer.

Fig. 4 has shown that the GFP in the mouse retina of biv vector transduction expresses.Fig. 4 A has described at 1 * 10 ofsubretinal injection 1 μ l⁵Transduction and expression in the tu two weeks back adult mice retina.Fig. 4 B has described 1 * 10 ofintravitreal injection 1 μ l⁵Transduction and expression in the tu two weeks back newborn mice.Fig. 4 C is the high power view of Fig. 4 B.

Fig. 5 has shown at the amphiblestroid smooth sample of the mouse of ocular administration BIV GFP carrier after one month, has demonstrated the high level expression of GFP in the RPE cell.

Fig. 6 has shown biv vector GFP expression in the monkey RPE cell after 10 weeks of using coding GFP.

Fig. 7 shows the biv vector former generation people RPE cell of effectively transduceing.Left figure shows the dyeing of RPE65 (the RPE specific proteins of 65KD).Middle figure and right figure show photopic vision field pattern and the fluorogram with the cell of GFP carrier transduction.

The biv vector of Fig. 8 code displaying Endostatin shows the usefulness that suppresses angiogenesis in the animal model of red angiogenesis.The picture left above has been described untreated eye.Lower-left two figure have described the eye with the control vector treatment, and the figure on right side has described the eye with BIV Endostatin vehicle treatment.Upside figure is a fluorescein angiography.Middle graph is amphiblestroid histologic section.Downside figure is the cross section of whole eye.

Fig. 9 has shown the carrier titre in the wash-out flow point of Sephacryl S 500-HR post and the curve of BSA level.For this research, the culture media supplemented of cmy vector has 2%FBS.Details is described among the embodiment 30.

Figure 10 shows that the biv vector of expressing T2-TrpRS has suppressed the angiogenesis in the damage from laser model.Figure 10 has shown the average-size from the new vessels zone of two formations.In the eye of T2-TrpRS (carboxyl-terminal fragment of tryptophan tRNA synzyme) vehicle treatment, the angiogenesis zone is obviously littler.

Figure 11 has described CCP and agglutinin complement pathway.Lectin pathway starts by mannose binding lectin (MBL) classical pathway by the C1 startup.C4bC2a is cut into C3 the protease of C3a and C3b and is called as C3 convertase.Similarly, C4bC2aC3b is cut into C5 C5a and C5b and is called as C5 convertase.C3a, C4a and C5a have inflammation character and attract phagocyte.C5b6-9 forms membrane attack complex (MAC), and this MAC creates the fenestra of killing infector but also damaging host cell.MASP is the relevant serine protease of mannan-binding lectin.

Figure 12 has described alternative complement pathway.This approach has low-level activity by spontaneous cutting and the composition ground of C3.In the presence of suitable surface, C3b combines with complement factor B (fB).Then this compound by Complement Factor D (fD) thus cutting generates C3bBb.Spontaneous dissociate (" decay ") of this compound in several minutes causes its inactivation, yet can produce the compound that can cut C3, i.e. C3 convertase by the stabilisation of properdin.Several factors that weaken complement pathway realize weaken (seeing the following form 1) to complement pathway by the decay of quickening C3 convertase and C5 convertase.Please note: thus C3b participates in C3 convertase to produce the extra positive feedback loop of C3b foundation shown in big arrow.C3bBb is a C3 convertase.C3bBbC3b is a C5 convertase.

Figure 13 has shown the expression of the fB that is derived from carrier in the human retina cell.With the carrier transduction ARPE cell based on BIV (RPE derived cell system) of coding fB construct, see embodiment 8.Medium from each transducer cell group is carried out Western analyze and detect fB.The 1st swimming lane, the purifying of 100ng is derived from the fB of human plasma; The 2nd～5 swimming lane, be respectively 40 μ l from medium with the cell of the carrier transduction of coding people wild type fB, fB3, fB2 and fB1.All swimming lanes are from identical glue.

Figure 14 has shown the structure of single-chain antibody, described single-chain antibody can be for example as heterologous gene by subclone to as in the transfer vector construct of Fig. 2 or from cellular expression.Leader (Leader) is the targeting sequencing that instructs secretion; V_LAnd V_HBe respectively variable light chain and heavy chain or comprise its partial C DR, connect by for example joint known in the art.In some embodiments, sequence of heavy chain can be placed the upstream of sequence of light chain.

Figure 15 has shown the result to the haemolysis test of people's wild type fB that is derived from carrier and fB dominant body (dominantnegative).Over against shining is the Erab (rabbit erythrocyte) that mixes with distilled water to produce 100% haemocylolysis.Purified fB albumen is represented the haemolysis by the serum of exhausting people fB of the fB in the purifying human plasma source that replenishes 500ng.Negative contrast expression is by only having exhausted the haemolysis of the human serum of fB.Five the bar posts in right side are represented by being added with the haemolysis with the serum of exhausting fB of the medium of the cell of following carrier transduction: the carrier of coding GFP, wild type people fB, fB1, fB2 or fB3.

Figure 16 has shown the transfer of lentiviral vector genome to rat aorta and mouse brain.Figure 16 A has shown with the transduction to the part rat aorta of the slow virus carrier in HIV source.Figure 16 B has shown with the gene transfer of BIV GFP carrier to mouse brain.Correlation technique is described among the embodiment 14.

Figure 17 has shown the result who substitutes the hemolytic activity test of complement pathway activity with various factor B mutant assessments.Y-axis has been showed the relative hemolytic activity of measuring by the hemoglobin level that is discharged into behind the erythrocyte splitting in the supernatant.X-axis is from left to right: 100% cracking over against photograph, the erythrocyte of cracking (RBC) in water; WT (wild type) hfB is with the blood plasma of 500ng purifying source people's factor B albumen (catalog number (Cat.No.) A408, Quidel, San Diego, CA) the additional human serum of exhausting factor B; Negative contrast, the RBC of incubation in isotonic saline solution (no erythrocyte cracking); Wild type hfB carrier is with the human serum of exhausting factor B with the culture media supplemented of the cell of the biv vector of encoding wild type factor B transduction; The GFP carrier is with the human serum of exhausting factor B with the culture media supplemented of the cell of the biv vector transduction of coding GFP; Mutant hfB1 carrier is with the human serum of exhausting factor B with the culture media supplemented of the cell of the biv vector of encoding mutant body fB1 transduction; Mutant hfB2 carrier is with the human serum of exhausting factor B with the culture media supplemented of the cell of the biv vector of encoding mutant body fB2 transduction; Mutant hfB3 carrier is with the human serum of exhausting factor B with the culture media supplemented of the cell of the biv vector of encoding mutant body fB3 transduction; GFP carrier+WT fB 1: 1, with ratio be 1: 1 with the medium of the cell of the biv vector transduction of coding GFP with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type factor B; Mutant hfB 1+WT fB 1: 1, with ratio be 1: 1 with the medium of the cell of the biv vector of encoding mutant body factor B 1 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type factor B; Mutant hfB2+WT fB 1: 1, with ratio be 1: 1 with the medium of the cell of the biv vector of encoding mutant body factor B 2 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type factor B; Mutant hfB3+WT fB 1: 1, with ratio be 1: 1 with the medium of the cell of the biv vector of encoding mutant body factor B 3 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type factor B; GFP carrier+WT fB 2: 1, with ratio be 2: 1 with the medium of the cell of the biv vector transduction of coding GFP with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type factor B; Mutant hfB1+WT fB 2: 1, with ratio be 2: 1 with the medium of the cell of the biv vector of encoding mutant body factor B 1 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type factor B; Mutant hfB2+WTfB 2: 1, with ratio be 2: 1 with the medium of the cell of the biv vector of encoding mutant body factor B 2 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type factor B; Mutant hfB3+WT fB 2: 1, with ratio be 2: 1 with the medium of the cell of the biv vector of encoding mutant body factor B 3 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type factor B.

Figure 18 has shown that assessment substitutes the result of the hemolytic activity test of complement pathway activity.Y-axis has been showed the relative hemolytic activity of measuring by the hemoglobin level that is discharged into behind the erythrocyte splitting in the supernatant.X-axis is from left to right: 100% cracking over against photograph, the RBC of cracking in water; Blank, the RBC of incubation in isotonic saline solution (no erythrocyte splitting); The mice serum (50ul) that in following sample (each 40ul), adds four times of dilutions: GFP carrier, the medium of the cell that the biv vector of using the GFP that encodes that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 1: 1 is transduceed; Mutant mfB1 carrier, the medium of the cell that the biv vector with encoding murine mutant fB1 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 1: 1 is transduceed; Mutant mfB2 carrier, the medium of the cell that the biv vector with encoding murine mutant fB2 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 1: 1 is transduceed; Mutant mfB3 carrier, the medium of the cell that the biv vector with encoding murine mutant fB3 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 1: 1 is transduceed; The GFP carrier, the medium of the cell that the biv vector with coding GFP that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 2: 1 is transduceed; Mutant mfB1 carrier, the medium of the cell that the biv vector with encoding murine mutant fB1 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 2: 1 is transduceed; Mutant mfB2 carrier, the medium of the cell that the biv vector with encoding murine mutant fB2 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 2: 1 is transduceed; Mutant mfB3 carrier, the medium of the cell that the biv vector with encoding murine mutant fB3 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 2: 1 is transduceed; The GFP carrier, the medium of the cell that the biv vector with coding GFP that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 1: 1 is transduceed; Mutant hfB1 carrier, the medium of the cell that the biv vector with coding people mutant fB1 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 1: 1 is transduceed; Mutant hfB2 carrier, the medium of the cell that the biv vector with coding people mutant fB2 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 1: 1 is transduceed; Mutant hfB3 carrier, the medium of the cell that the biv vector with coding people mutant fB3 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 1: 1 is transduceed; The GFP carrier, the medium of the cell that the biv vector with coding GFP that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 2: 1 is transduceed; Mutant hfB1 carrier, the medium of the cell that the biv vector with coding people mutant fB1 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 2: 1 is transduceed; Mutant hfB2 carrier, the medium of the cell that the biv vector with coding people mutant fB2 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 2: 1 is transduceed; Mutant hfB3 carrier, the medium of the cell that the biv vector with coding people mutant fB3 that mixes with medium with the cell of the biv vector transduction of encoding wild type mouse factor B with ratio 2: 1 is transduceed.

Figure 19 has shown that assessment substitutes the hemolytic activity test of complement pathway activity.Y-axis has been showed the relative hemolytic activity of measuring by the hemoglobin level that is discharged into behind the erythrocyte splitting in the supernatant.X-axis is from left to right: 100% cracking over against photograph, the RBC of cracking in water; Blank, the RBC of incubation in isotonic buffer solution (no erythrocyte splitting); Medium with the cell of BIV GFP carrier transduction; WT hfB carrier is with the human serum of exhausting factor B with the culture media supplemented of the cell of the biv vector of encoding wild type factor B transduction; Mutant hfB1 carrier is with the human serum of exhausting factor B with the culture media supplemented of the cell of the biv vector of encoding mutant body fB1 transduction; Mutant hfB2 carrier is with the human serum of exhausting factor B with the culture media supplemented of the cell of the biv vector of encoding mutant body fB2 transduction; Mutant hfB3 carrier is with the human serum of exhausting factor B with the culture media supplemented of the cell of the biv vector of encoding mutant body fB3 transduction; GFP carrier+1: 1wWT hfB, with ratio be 1: 1 with the medium of the cell of the biv vector transduction of coding GFP with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type people factor B; Mutant mfB 1+1: 1wWT hfB, with ratio be 1: 1 with the medium of the cell of the biv vector of encoding murine mutant factor B 1 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type people factor B; Mutant mfB2+1: 1wWT hfB, with ratio be 1: 1 with the medium of the cell of the biv vector of encoding murine mutant factor B 2 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of coding people wild type factor B; Mutant mfB3+1: 1wWT hfB, with ratio be 1: 1 with the medium of the cell of the biv vector of encoding murine mutant factor B 3 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of coding people wild type factor B; GFP carrier+2: 1wWT hfB, with ratio be 2: 1 with the medium of the cell of the biv vector transduction of coding GFP with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type people factor B; Mutant mfB1+2: 1wWT hfB, with ratio be 2: 1 with the medium of the cell of the biv vector of encoding murine mutant factor B 1 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type people factor B; Mutant mfB2+2: 1wWT hfB, with ratio be 2: 1 with the medium of the cell of the biv vector of encoding murine mutant factor B 2 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of coding people wild type factor B; Mutant mfB3+2: 1wWT hfB, with ratio be 2: 1 with the medium of the cell of the biv vector of encoding murine mutant factor B 3 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of coding people wild type factor B; GFP carrier+4: 1wWT hfB, with ratio be 4: 1 with the medium of the cell of the biv vector transduction of coding GFP with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type people factor B; Mutant mfB1+4: 1wWT hfB, with ratio be 4: 1 with the medium of the cell of the biv vector of encoding murine mutant factor B 1 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of encoding wild type people factor B; Mutant mfB2+4: 1wWT hfB, with ratio be 4: 1 with the medium of the cell of the biv vector of encoding murine mutant factor B 2 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of coding people wild type factor B; Mutant mfB3+4: 1wWT hfB, with ratio be 4: 1 with the medium of the cell of the biv vector of encoding murine mutant factor B 3 transduction with the additional human serum of exhausting factor B of mixture of the medium of the cell of the biv vector transduction of coding people wild type factor B.

Figure 20 has shown that assessment substitutes the hemolytic activity test of complement pathway activity.Y-axis has been showed the relative hemolytic activity of measuring by the hemoglobin level that is discharged into behind the erythrocyte splitting in the supernatant.X-axis is from left to right: 100% cracking over against photograph, the RBC of cracking in water; Negative contrast, the RBC of incubation in isotonic saline solution (no erythrocyte splitting); The GFP carrier, the medium (40ul) of the cell that the biv vector with coding GFP that mixes with the pig serum (50ul) of twice dilution is transduceed; Wild type hfB carrier, the medium (40ul) of the cell that the biv vector with the encoding wild type factor B that mixes with the pig serum (50ul) of twice dilution is transduceed; Mutant hfB1 carrier, the medium (40ul) of the cell that the biv vector with coding people mutant fB1 that mixes with the pig serum (50ul) of twice dilution is transduceed; Mutant hfB2 carrier, the medium (40ul) of the cell that the biv vector with coding people mutant fB2 that mixes with the pig serum (50ul) of twice dilution is transduceed; Mutant hfB3 carrier, the medium (40ul) of the cell that the biv vector with coding people mutant fB3 that mixes with the pig serum (50ul) of twice dilution is transduceed; The GFP carrier, the medium (40ul) of the cell that the biv vector with coding GFP that mixes with the pig serum (50ul) of four times of dilutions is transduceed; Wild type hfB carrier, the medium (40ul) of the cell that the biv vector with the encoding wild type factor B that mixes with the pig serum (50ul) of four times of dilutions is transduceed; Mutant hfB1 carrier, the medium (40ul) of the cell that the biv vector with coding people mutant fB1 that mixes with the pig serum (50ul) of four times of dilutions is transduceed; Mutant hfB2 carrier, the medium (40ul) of the cell that the biv vector with coding people mutant fB2 that mixes with the pig serum (50ul) of four times of dilutions is transduceed; Mutant hfB3 carrier, the medium (40ul) of the cell that the biv vector with coding people mutant fB3 that mixes with the pig serum (50ul) of four times of dilutions is transduceed; The GFP carrier, the medium (40ul) of the cell that the biv vector with coding GFP that mixes with the pig serum (50ul) of six times of dilutions is transduceed; Wild type hfB carrier, the medium (40ul) of the cell that the biv vector with the encoding wild type factor B that mixes with the pig serum (50ul) of six times of dilutions is transduceed; Mutant hfB1 carrier, the medium (40ul) of the cell that the biv vector with coding people mutant fB1 that mixes with the pig serum (50ul) of six times of dilutions is transduceed; Mutant hfB2 carrier, the medium (40ul) of the cell that the biv vector with coding people mutant fB2 that mixes with the pig serum (50ul) of six times of dilutions is transduceed; Mutant hfB3 carrier, the medium (40ul) of the cell that the biv vector with coding people mutant fB3 that mixes with the pig serum (50ul) of six times of dilutions is transduceed.

Figure 21 has shown the cutting of factor D to people's factor B of C3b dependence.The Western trace of total length factor B or factor B cleaved products (Bb and Ba) detects.The 1st swimming lane is as the purifying people factor B albumen over against photograph; The 2nd swimming lane is with the medium by the cell of the biv vector transduction of coding GFP of factor D incubation; The 3rd swimming lane is with the medium by the cell of the biv vector transduction of coding people wild type factor B of factor D incubation; The 4th swimming lane is with the medium by the cell of the biv vector transduction of coding people mutant factor B 1 of factor D incubation; The 5th swimming lane is with the medium by the cell of the biv vector transduction of coding people mutant factor B 2 of factor D incubation; The 6th swimming lane is with the medium by the cell of the biv vector transduction of coding people mutant factor B 3 of factor D incubation; The 7th swimming lane, molecular weight marker; The 8th swimming lane is with the medium by the cell of the biv vector transduction of coding GFP of C3b and factor D incubation; The 9th swimming lane is with the medium by the cell of the biv vector transduction of coding people wild type factor B of C3b and factor D incubation; The 10th swimming lane is with the medium by the cell of the biv vector transduction of coding people mutant factor B 1 of C3b and factor D incubation; The 11st swimming lane is with the medium by the cell of the biv vector transduction of coding people mutant factor B 2 of C3b and factor D incubation; The 12nd swimming lane is with the medium by the cell of the biv vector transduction of coding people mutant factor B 3 of C3b and factor D incubation.The reactant mixture of each sample is described in the table 2.

Figure 22 has shown that factor B and C3b are in conjunction with test.With C3b, factor D and factor B (wt or mutant) incubation together in reactant mixture.With polyclone anti-factor B antiserum this reactant mixture is carried out immunoprecipitation, separate, and analyze by the Western engram analysis with anti-C3b polyclonal antiserum by SDS-PAGE.The 1st swimming lane is as over against the purifying C3b albumen of photograph (is C3 beta chain with the C3b copurification than the band of below); The 2nd swimming lane is with the medium with the cell of the biv vector transduction of coding GFP of C3b and factor D incubation; The 3rd swimming lane is with the medium with the cell of the biv vector transduction of coding people wild type factor B of C3b and factor D incubation; The 4th swimming lane is with the medium with the cell of the biv vector transduction of coding peoplemutant factor B 3 of C3b and factor D incubation; The 5th swimming lane is with the medium with the cell of the biv vector transduction of coding peoplemutant factor B 2 of C3b and factor D incubation; With the 6th swimming lane, with the medium with the cell of the biv vector transduction of coding peoplemutant factor B 1 of C3b and factor D incubation.

Figure 23 has shown the test to people's complement factor B and factor D combination.Experimental detail can find among the embodiment 21 below.The 1st swimming lane, negative contrast is reacted not existing under C3b, factor D and the factor B situation; The 2nd swimming lane is with the medium with the cell of the biv vector transduction of coding GFP of C3b and factor D incubation; The 3rd swimming lane is with the medium with the cell of the biv vector transduction of coding people wild type factor B of C3b and factor D incubation; The 4th swimming lane, molecular weight marker; The 5th swimming lane is with the medium with the cell of the biv vector transduction of coding peoplemutant factor B 3 of C3b and factor D incubation; The 6th swimming lane is with the medium with the cell of the biv vector transduction of coding peoplemutant factor B 2 of C3b and factor D incubation; The 7th swimming lane is with the medium with the cell of the biv vector transduction of coding peoplemutant factor B 1 of C3b and factor D incubation; With the 8th swimming lane, as purifying people factor B over against photograph.

The hemolytic activity test that Figure 24 utilizes assessment to substitute the complement pathway activity has shown the inhibition of anti-people's factor B monoclone antibody to people's alternative complement pathway activity.Y-axis has been showed the relative hemolytic activity of measuring by the hemoglobin level that is discharged into behind the erythrocyte splitting in the supernatant.X-axis is from left to right: 100% cracking over against photograph, the RBC of cracking in water; Purifying hfB albumen is with the additional human serum of exhausting factor B of the purifying people factor B albumen of 500ng; Negative contrast, RBC is incubation (no erythrocyte splitting) in isotonic saline solution; The mAb 0.4 μ g of anti-hfB; The human serum of exhausting factor B that replenishes with the mixture of the purifying people factor B albumen of the mAb (0.4 μ g) of anti-hfB and 500ng; The mAb 0.8 μ g of anti-hfB; The human serum of exhausting factor B that replenishes with the mixture of the purifying people factor B albumen of the mAb (0.8 μ g) of anti-hfB and 500ng; The mAb1.6 μ g of anti-hfB, the human serum of exhausting factor B that replenishes with the mixture of the purifying people factor B albumen of the mAb (1.6 μ g) of anti-hfB and 500ng; The mAb 2.4 μ g of anti-hfB; The human serum of exhausting factor B that replenishes with the mixture of the purifying people factor B albumen of the mAb (2.4 μ g) of anti-hfB and 500ng; Control mice IgG 0.4 μ g, the human serum of exhausting factor B that replenishes with the mixture of the purifying people factor B albumen of control mice IgG (0.4 μ g) and 500ng; Control mice IgG 0.8 μ g, the human serum of exhausting factor B that replenishes with the mixture of the purifying people factor B albumen of control mice IgG (0.8 μ g) and 500ng; Control mice IgG 1.6 μ g, the human serum of exhausting factor B that replenishes with the mixture of the purifying people factor B albumen of control mice IgG (1.6 μ g) and 500ng; Control mice IgG 2.4 μ g, the human serum of exhausting factor B that replenishes with the mixture of the purifying people factor B albumen of control mice IgG (2.4 μ g) and 500ng.

Figure 25 has shown the analysis to people'sfactor B 3 albumen.Figure A has shown the silver dyeing of people'sfactor B 3 albumen of affinity purification: the 1st swimming lane, molecular weight marker; The 2nd swimming lane: from the elution samples of first flow point; The 3rd swimming lane is from second flow point of combination and the elution samples of the 3rd flow point.Figure B has shown the distribution of the Western engram analysis of people'sfactor B 3 albumen and swimming lane identical with figure A.

Figure 26 has shown that assessment substitutes the hemolytic activity test of complement pathway activity.Y-axis has been showed the relative hemolytic activity of measuring by the hemoglobin level that is discharged into behind the erythrocyte splitting in the supernatant.X-axis is from left to right: 100% cracking over against photograph, the RBC of cracking in water; Blank, RBC is incubation (no erythrocyte splitting) in isotonic saline solution; Wild type factor B albumen (50ng, 100ng, 200ng and 500ng) is with the additional human serum of exhausting factor B of wild type people's factor B (Quidel) of 50ng, 100ng, 200ng and 500ng; Wild type factor B albumen+factor B 3 albumen add the human serum of exhausting factor B that the mixture of wild type people's factor B of 0ng, the 200ng of Quidel or 500ng replenishes with themutant factor B 3 of the affinity purification of 40 μ l.

Figure 27 has shown that the GFP in the mouse retina expresses and C3 dyeing.Figure 27 A and 27B: as described in embodiment 27, use the GFP carrier.After two weeks, prepare smooth sample and check with fluorescence microscope.Figure 27 C and 27D: as described in embodiment 27, use empty carrier and hfB3 carrier.After two weeks, near foveal region of retina, carry out laser photocoagulation.After 20 hours, collect retina, the C3 sediments is dyeed, and check with fluorescence microscope.

Figure 28 has shown the carrier titre from each wash-out flow point of Sephacryl S 500-HR post.For this research, not to the culture media supplemented FBS of cmy vector.Details is described among the embodiment 32.

Figure 29 has described following plasmid.Figure 29 A is pAVTrGP038 (SEQ ID NO:19).Figure 29 B is pAVTrREV039 (SEQ ID NO:20).Figure 29 C is pAVTrGP64-040 (SEQ IDNO:21).Figure 29 D is pAVT001 (SEQ ID NO:22).Figure 29 E is pAVTGFP006 (SEQID NO:23).

Embodiment

Unless otherwise noted, practice of the present invention belongs to employing the routine techniques of cell biology, molecular biology, cell culture and the virology etc. of those skilled in the art's technology.These technology fully are disclosed in the existing document, for example, " Molecular Cloning, A Laboratory Manual " writes in institutes such as Sambrook, Fritsch and Maniatis, the 2nd edition, Cold Spring HarborLaboratory Press (1989); " Cell Biology, the A LaboratoryHandbook " of Celis J.E., Academic Press, J.of Virol.Methods such as Inc. (1994) and Bahnson, 54:131-143 (1995).

Term " complement-mediated " is meant process or the disease that relates to complement.Usually, the disease of " complement-mediated " or the patient's condition are the so a kind of disease or patient's condition: wherein complement activity is the basic cause of the disease or the patient's condition and the degree that the inhibition or the blocking-up of complement activity alleviated the described disease or the patient's condition.This paper has described the example of the disease or the patient's condition of many complement-mediated.

Term " complement protein " or " complement pathway composition " are albumen or its acceptors of complement system.Complement protein is one group of about 35 kinds of interactional albumen and the glycoprotein that is found in all vertebrates.Complement protein can be soluble or be in (Sim and Tsiftsoglou, Biochemical Society Transactions (2004) 32 (1): 21-27) on the cell surface.In addition, the adjusting memebrane protein of protecting host cell not attacked by unexpected or undesirable complement in addition.Complement protein can be albumen (for example, the albumen (for example, factor B) of performance function C2) or in alternative route of performance function in classical pathway.Having 6 kinds of complement proteins at least is protease.For example, included albumen is complement protein in the following exemplary lists: C1q, C1r, C1s, C2-9, factor B, factor D, factor H, factor I, CR1, CR2, CR3, CR4, properdin, C1 (Inh), C4bp, MCP, DAF, CD59 (MIRL) and HRF.

Exchanging the term used herein " wild type " that uses with " natural " relates to by the naturally occurring albumen of mammalian genes group coding, naturally occurring nucleic acid, naturally occurring individuality or animal or the like.

" complement protein variant ", " complement protein mutant " or " complement protein analog " are used interchangeably and refer to the structural derivative of parent's albumen, and this structural derivative may not keep all character of natural (naturally occurring) parent albumen or it compares the character with at least a change with natural parent's albumen.Can carry out the amino acid replacement by natural acid sequence, replace, lack and/or add and produce analog or variant for albumen.Described replacement or insertion are usually directed to naturally occurring amino acid, but also can comprise synthetic or unconventional amino acid.In some embodiments, by making protein mutation (for example, making its nucleic acid mutation of coding) produce analog or variant.The amino acid sequence that analog can keep natural parent's albumen of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% usually (for example; compare amino acid sequence identity with naturally occurring parent's albumen with described percentage; described amino acid sequence identity is determined on the length of whole parent's albumen, perhaps determines at the special domain of described parent's albumen or partly in some embodiments).Analog also comprises the fragment of total length analog, described fragment comprise the part (for example, at least 30,50,70,100,150,200,300,400,500,600 or 700 amino acid or comprise the subgroup in territory of described analog or territory) of amino acid sequence and kept parent's albumen or total length analog more than one biologically active or suppressed more than one these biologically actives.

Be meant the specific amino acids in this specific protein during the amino acid of term " correspondence " in referring to specific protein and also refer to provide amino acid in the relevant or similar protein with the function of described protein similar.For example, in the amino acid in possible finder's complement factor B and the muroid complement factor B or the amino acid in the people's of factor B the allelic variation body corresponding, this determines by comparing two amino acid sequences usually.For example, those skilled in the art can for example use blast program two correlated serieses of comparison (as SEQ ID NO:2 and SEQ ID NO:16) to determine corresponding amino acid.In addition, can be for example by the motif in relevant or the incoherent albumen (for example, protease cutting motif) be compared to determine corresponding amino acid.Also can use such comparison to draw the consensus sequence in target proteins or its territory.

As used herein, term " gene " is often referred to the code area to albumen.Yet, term " gene " also obviously refers to and the element (for example, controlling element) that is connected as code area navigabilities such as promotor, enhancer, splice site (acceptor site and/or donor site), polyadenylation signal, intron, 5 ' non-translational region, 3 ' non-translational regions under some environment of this paper.

As used herein, being called genetically modified " interested gene " sometimes is heterologous gene or alien gene for the source of nucleic acid carrier or vector construction body.Thereby for example in the situation of biv vector, interested gene is not the BIV gene usually.In some embodiments, interested gene is a therapeutic gene.

Term " packaging sequence " is that viral nucleic acid is packaged into necessary sequence in virion, virion or the virus-like particle.For example, in BIV, packaging sequence is usually located at the zone between the upstream of 5 ' main donor splicing site and gag gene.Support according to the present invention can comprise the packaging sequence corresponding to other virus (for example, as slow viruss such as HIV-1, HIV-2 or SIV).Packaging sequence can comprise 1000 nucleotide of as many as or few to 50 nucleotide.Those of ordinary skills can easily determine the size in packaging sequence district.

Term " medicine is acceptable " is meant through management organization's approval of federal government or state government or at American Pharmacopeia or other human pharmacopeia that is used for of generally acknowledging and lists.

" treatment benefit " may not be the healing to the specified disease or the patient's condition (comprising any disease as herein described or the patient's condition), but contained the result who generally includes following situation most: alleviate the described disease or the patient's condition, eliminate the described disease or the patient's condition, reduce one or more symptoms with the described disease or the patient's condition, prevention or alleviate the secondary disease or the patient's condition that the appearance by the primary disease or the patient's condition causes, weaken the possibility of the patient's condition or advancing of disease, weaken the seriousness of the disease or the patient's condition, change the proterties of the disease or the patient's condition, shorten the course of disease of the disease or the patient's condition, slow down or ward off disease or the progress or the deterioration of the patient's condition, and/or prevent the described disease or the patient's condition.

" conversion " is intended to comprise any way with in external source, allos or the external nucleic acid introducing cell.This can be as known in the art like that by using nucleic acid itself, plasmid (transfection), virus (infecting or transduction), synthetic carrier molecule or the like to carry out.Thereby through cell transformed is to handle the cell that carries interested external nucleic acid with variety of way arbitrarily.These cells can be the stem cell and the embryonic stem cells of somatic cell, non-embryonic stem cells.

" vector construction body " or " carrier sequence " or " transfer vector " are meant the assembly of the expression that can instruct nucleotide sequence, transgenosis, albumen or therapeutic expression product.In some embodiments of the present invention, the vector construction body can comprise and can start the 5 ' sequence of transcribing (comprising the promoter region that navigability connects); From virus genomic dna fragmentation; And/or come the packaging sequence of the virus of slow virus or retrovirus etc. freely.In one embodiment, the invention provides the biv vector construct, described biv vector construct comprises: from the genomic dna fragmentation of BIV, be used for RNA is packaged into packaging sequence, first promotor that is connected with described dna fragmentation navigability and the transgenosis that is connected with the second promotor navigability of virion.In one embodiment, the packaging sequence of biv vector construct is the BIV packaging sequence.

Target molecules

" target molecules " is the molecule that can participate in approach, thus they self or its activity can be regulated the adjusting that causes described approach.Target molecules may not be participated in approach directly, and for example, it may have the activity that influences another second molecule, and the molecule of described approach is participated in this second molecule and then influence directly.In some embodiments of the present invention, can increase, reduce or keep the quantity and/or the activity of target molecules.This can by but be not limited to regulate the expression of described target molecules or make some target molecules at least go more than one biologically active stable or that eliminate some target molecules at least, make described molecule isolate (sequester) and/or change described target molecules to finish.In some embodiments, when described target molecules is albumen or nucleic acid, can come transformant (for example, external or in vivo) to finish adjusting by at least a nucleic acid to the expression of described target molecules with the described target molecules of coding.In some embodiments, can make target proteins go stable or reduce its active binding molecule by employing and make described target proteins unstable or abrogate or reduce its activity.In some embodiments, thus can adopt and combine the stable binding molecule that goes that causes described target proteins with target proteins.In some embodiments, can use the protease that cuts described target proteins to make to the small part target molecules goes to stablize or eliminate to the small part target molecules.In some embodiments, protease in target molecules 1,2,3,4,5,6,7,8,9,10 or more site are cut described target molecules.Some embodiment of the present invention suppresses target molecules.Inhibitor of the present invention comprises but is not limited to binding molecule as comprising that immunoglobulin fragment is (as Fab, F (ab ')₂And F_v) antibody molecule (and homologue, analog and modified forms or derivative form), little molecule, peptide, oligonucleotides, fit, peptide mimics and organic compound.

Complement system can mediate the chain reaction of the assembling of proteolysis and albumen composition, for example causes eliminating the chain reaction of the assembling of the proteolysis of invading microorganism and albumen composition.Three kinds of activated pathway (classical pathway, lectin pathway and alternative route) and lytic pathway are regulated these incidents.

In classical pathway as shown in figure 11, the immunoglobulin in the collagen subcomponent C1q of first composition (C1) and the immune complex combines, and its relevant serine protease (C1r and C1s) obtains activating.Cutting to C4 and C2 has subsequently started this complement cascade, and then C3 activates.Gained C3b fragment is not only served as opsonin but also has been caused the formation of membrane attack complex (MAC) in the lytic pathway.In the innate immunity, form compound by identification molecule (agglutinin) with the serine protease of the relevant serine protease of mannose binding lectin by name (MBL) (MASP), described compound has activated C4 and C2 when combining via the lip-deep sugar of lectin pathway and microorganism.This is not combined in when having immunoglobulin and takes place.The identification molecule of the lectin pathway of finding in jawed vertebrates is MBL and fiber gelatinized protein (ficolin), and both features all are similarly there is the collagen-like territory with C1q and the sugar that has the common binding specificity of GlcNAc combines the territory.MASP enjoys identical tract tissue with C1r/C1s and forms the subtribe of serine protease.

Agglutinin complement pathway and the CCP in the adaptive immunity in the innate immunity are closely related, for example, and for their 26S Proteasome Structure and Function of composition.Usually these two approach are all started by following compound, and described compound is made up of with the serine protease of the relevant serine protease of mannose binding lectin (MBL) (MASP)/C1r/C1s family collagen.Classical pathway occurs after lectin pathway by inference.

As shown in figure 12, when C3b (or C3i) and (for example, microorganism) cell and other surface composition in conjunction with the time, the activation that substitutes complement pathway begins.C3b also can with the IgG antibodies.Thereby alternative route protein factor B and C3b merge formation C3bB then.Factor D then the factor B of combination be divided into Bb and Ba, form C3bBb.Properdin combines with Bb to form C3bBbP then, and C3bBbP is bringing into play the function that hundreds of C3 molecule enzymatics can be cut into the C3 convertase of C3a and C3b usually.Substituting complement pathway now is activated.Some C3b combines with some C3bBb immediately to form C3bBbC3b, and C3bBbC3b is the C5 convertase that the C5 molecule can be divided into C5a and C5b.

Because C3b is free in the blood plasma, so it can combine with host cell or pathogene surface.In order to prevent that complement activation to carrying out on the host cell, having several inhomogeneous adjusting albumen can interrupt the complement activation process.Complement receptor 1 (CR1 or CD35) and DAF (being also referred to as CD55) combine with C3b on cell surface and even Bb can be removed from the C3bBb compound that has formed with the factor B competition.When being cut into its inactive form iC3b with C3b, the plasma proteinase that is called factor I also can prevent the formation of C3 convertase.Factor I combines albumen co-factor such as CR1 and proteolysis film co-factor (MCP or CD46) etc. and together works with C3b.Another Complement Regulatory Protein is factor H, the competition of factor H and factor B, Bb is replaced, serves as the co-factor of factor I or preferentially combines with the C3b that is attached to vertebrate cells from invertase.

Target molecules includes but not limited to composition or its variant of immunology approach, as the composition of CCP; Substitute the composition of complement pathway or the composition of agglutinin complement pathway.In some embodiments, can be regulated and/or be acted on more than a kind of target molecules.In some embodiments, the composition of alternative complement pathway is selected from the group that is grouped into by C3, C3a, C3b, C3bB, fB (factor B), fD (factor D), C3bBb, C3bBbC3b, C5, C5a, C5b, C6, C7, C8, C9, C5b6-9, MAC (membrane attack complex) and fragment thereof and their one-tenth.In some embodiments, the composition of CCP is selected from the group of being made up of C1r, C1q, C1s, C4, C4b, C4a, MBL, MASP, C2, C2b, C4bC2a, C3, C3b, C3a, C4bC2aC3b, C5, C5a, C5b, C6, C7, C8, C9, C5b6-9 and MAC.

Start or promote that the target molecules of inflammation will be by expressing and/or benefit will be brought into play in active reduction, and comprise properdin, TGF β, complement factor B (people's complement factor B, accession number NP001701, Kavanaugh etc., Mol.Imm.43 (7) 856,2006; Mouse complement factor B, accession number NP 032224m, Bora etc., J.Imm.177 (3) 1872,2006), Complement Factor D and comprise other factor of the complement system of C2, C3, C4, C5, C6, C7, C8 and C9.Thereby, interested transportation means can express specific antigen for example in conjunction with polypeptide, soluble recepter, inhibition nucleic acid, ribozyme, fit, catalytic antibody, have the molecule of aminoacid replacement, they can disturb as prevent with the butt joint of acceptor, making combine irreversible, remove function such as enzymic activity.Select as another kind, can use can with as target molecules such as acceptor compete but the molecule that can not effectively activate or promote inflammation as competitive inhibitor so that weaken inflammation.In addition, can use to have the active of change or do not have (shortage) active complement factor in practice of the present invention, described activity can be one or more given activity or the function of any molecule.Change and to mean and be different from activity or the function that under normal environmental condition, obtains, for example operation or present higher levels of activity or lower level activity under higher activity.Also can application in practice other reduction known in the art express or active method.

Complement factor usually is multi-functional.Thereby, can think that these molecules have a plurality of territories.For example, complement factor may have function or the active territory that has identification and combination; By the territory of another molecular recognition, can react, change or cut this site or its close region; May have as enzymic activity bioactive territories such as (as proteinase activities) or the like.When the function in any one or a plurality of described territories was changed, the biologically active of complement factor may be weakened or be eliminated, and the change in a territory may also may not can influence the normal function in another territory.Can be by for example in described territory, replacing, inserting and/or lack amino acid and measure desired character and realize change to the albumen territory.Even the variant of the function of change still can the using system method be located and is obtained to have in the position the unknown in described territory.

Approach of the present invention is regulated son and molecule

Can the many molecules of target in the treatment of complement-mediated disease (as the complement-mediated disease relevant) with illness in eye.To alleviate or to improve inflammation or do not allow inflammation to continue as target, the present invention partly relates to the molecule part or systematically transports the described result of realization.Thereby In some embodiments of the present invention, molecule can be to startup, assist or promote the expression or the active molecule that suppresses of the target molecules of inflammation, or make the expression of the target molecules that weakens inflammation or the molecule of increased activity.

In some embodiments, the present invention has considered that the activity of any or multiple composition by regulating approach as herein described comes the adjusting approach and use any method in order to the activity of regulating pathway components as herein described.

Immunology approach and process have the patient's condition (for example disease) that helps animal development, keep and/or suppress.The present invention partly provides the method for for example regulating (for example, enhancing, raising, inhibition, minimizing or generality are kept) immunology approach in external or body.Some embodiment of the present invention can be used for studying immunology approach, research relevant disease state, exploitation to the treatment of morbid state, set up morbid state (for example, exploitation is as the disease model in the animals such as mouse or rat) or be used for screening of medicaments animal.

In some embodiments, target molecules is the composition of complement pathway as herein described for example.Some embodiment of the present invention relates to CCP; Substitute the adjusting of complement pathway or agglutinin complement pathway.

Some embodiment of the present invention can reduce, improves or prevent inflammation and/or complement activity.Some embodiment of the present invention has been regulated complement function or activity.Since complement by many factors that work with cooperating type, many adjustings that on classics, is not counted as complement or control complement factor exist and the active molecule and the factor formed, any described complement factor or complement are regulated molecule can serve as entrance of the present invention or target molecules with as the mode that influence complement activity and function (for example, comparing enhancing or reduction complement activity with benchmark).

Can be for example by directly influencing the albumen in the complement pathway expression (for example, transcribe and/or translate) or biologically active, perhaps by directly influence in albumen and the complement pathway protein combination or by alternative route (front or negatively) thus the ability that complement activation is made contributions is realized the inhibition to complement pathway of the present invention.In some embodiments, the expression of albumen is meant the translation of transcribing of described albumen or described albumen.Therefore, some method of the present invention (for example, by using the reagent that suppresses described protein expression and/or animal being carried out genetic modification so that it has the protein expression of minimizing) can suppress that albumen is carried out natural expressed proteins and transcribe and/or translate (for example, in animal).In another embodiment, any (detectable) of measuring that this paper is defined as the inhibition of complement pathway the activity of described approach (for example reduces, reduce, reduce or suppress), for example by substituting expression and/or bioactive any minimizing of measuring of the albumen in the complement pathway.In another embodiment, the present invention relates to the expression of the factor (as complement factor H, DAF or the like) of reduction complement activity and/or the enhancing of function.

In some embodiments, interested carrier can be expressed known complement inhibitor, as C1 esterase inhibitor (Kirschfink ﹠amp; Molines, Expert Opin.Pharmacother.2,1073,2001) or compstatin (Ile-Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys-Thr-NH2, (Disulfide bond), Nilsson etc., Blood, 92,1661,1998).Regulate complement system and be other target molecules of substituting target interested comprise HtrA1 (Oka etc., Development 131,1041,2003; Suppress the transduction of TGF signal beta, the stimulant that known C3 and factor B are expressed) and CRP.

In some embodiments, weakened, reduced, improved, strengthened or kept the activity of target molecules.This can utilize various technology to realize.In some embodiments, binding molecule (for example, albumen, part, acceptor or antibody) combines with target molecules.In some embodiments, a kind, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 9 kinds, 10 kinds or more kinds of binding molecules combine with target molecules.In some embodiments, binding molecule combines with target proteins and suppresses target proteins, and described inhibition to target proteins is for example by the site on the active relevant target molecules of sealing, partially enclosed and target molecules or compete combination to described site; By competing combining to target molecules with part (for example native ligand); Undertaken by making target molecules be degraded (for example, utilize and comprise the directed degraded of protein-bonded macrophage that partly waits the macrophage bound fraction as Fc).

As what discussed in the method in the practice of the present invention, the coded sequence of target molecules (for example participate in pathways of inflammation or regulate the target molecules of pathways of inflammation) no matter be nucleic acid or albumen can both be changed and/or operate so that: the expression product that produces maneuverability or non-maneuverability; Produce the dominant expression product; That generation has more maneuverability or have a more highly active expression product; Thereby or produce wherein that its specific function can be operated the molecule with activity reduction or that eliminate.Have in conjunction with or be to be used for selectivity to reduce active suitable targets to the target molecules (having the territory of the multiple function of tool or the molecule of part charge) of connection function, enzymatic function or the like as those.For example, thus can be changed do not combine with natural receptor or with its irreversible target molecules that combines.Make enzymic activity forfeiture or weaken thereby can operate target molecules.Can carry out this variation with known materials in this area and method, for example, as introducing aminoacid replacement by rite-directed mutagenesis.Some illness that starts from treatment of the present invention may be caused or be had the symptom of inflammation by inflammation, and known in this area, described inflammation is characterised in that release or activation, vascular exosmosis, leukocyte infiltration and/or the tissue damage of inflammatory cytokine.Thereby, for purposes of the present invention, the disease that presents inflammation be with inflammation as the disease of the cause of disease or in the course of disease with the feature of inflammation disease as symptom or performance.

In another embodiment, thus can use molecule to capture and isolate target molecules and prevent that their from interacting and carry out its normal biological function with binding ability.Thereby, for example, can come the local specific molecular of carrying or expressing the soluble complement acceptor or combine, thereby described molecule through combination is minimized its short scorching active performance with interested vector construction body with the inflammation-induced thing.

For example, but the target molecules of inflammation-inhibiting will be brought into play benefit by the expression and/or the activity that strengthen, and comprise for example complement receptor 1 (CR1, also claim CD35, can combine that the soluble form of CR1 is at Weisman etc. with C3b, Science 249,146, describe to some extent in 1990); C4 is in conjunction with albumen and bound fraction thereof; Acrasin, S albumen and homologous restriction factor (HRF), this three suppresses the formation of MAC; The relevant albumen of complement receptor 1/gene y (Crry); Complement factor I (decomposing C3b); Complement factor H (inhibiting factor B combines with 3b's); DAF (also claim CD55, C3 convertase dissociates); Membrane cofactor protein (MCP also claims CD46, is the co-factor of complement factor I); Prevent to form the reactive cracking film inhibitor (also claiming MIP or CD59) of membrane attack complex (MAC); And FHL-1.Thereby interested mode of movement can be introduced the additional copy or the expression copy of the nucleic acid of expressing protein (for example above-mentioned those albumen) in cell (for example eye cell); Perhaps can such as by insert through enhancer that navigability connects or inducible promoter introduce make in the mode of expression enhancing of source coding sequence, for example see United States Patent (USP) the 5th, 272, No. 071 and the 6th, 303, No. 379.Can put into practice the mode and the method for other acquisition expression as known in the art like that.The present invention has also considered the conveying of albumen.Therefore, the described albumen of direct conveying itself of part for example and/or system has also been considered in any discussion to expressing protein or peptide.

According to the present invention, the complement protein analog is provided, described complement protein analog is compared structurally with naturally occurring complement protein and is modified, thereby as proteolytic activity, stability, binding affinity, target specificity, to the sensitivity of regulating albumen, aspect more than one character in proteoclastic sensitivity and the co-factor requirement etc., have functionalized modification.The modification of these character of complement protein analog can directly or indirectly influence their complement-mediated activity.These analogs can be used for regulating complement system.Can make the proteolytic activity, stability of complement protein, to the binding affinity of target, increase or reduce to the sensitivity of regulating albumen and/or to proteoclastic sensitivity.Can widen or constriction substrate specificity and/or can make stricter or more not strict or with its abolishment the requirement of co-factor.In some embodiments, produce the complement protein of modification by the sudden change in the zone of the above-mentioned character of control.

In some embodiments, the present invention relates to for example analog and/or the inhibitor of C1r, C1s, factor B, factor D, factor I, C3b and/or C2.The present invention also provides method and the coded polynucleotide that is used for preparing these analogs, the amino acid sequence that comprises described sudden change, in the treatment of associated conditions as the pharmaceutical composition of these analogs of complement therapeutic agent and use these analogs as the diagnostic method of reagent the reference material of the competitive ELISA of complement protein in serum or the tissue sample etc. (for example, as).

The sudden change that can modify complement protein comprises such as at short consensus repeat (SCR), vWF ELISA (von Willebrand Factor, vWF) in territory and/or the protease domain or with substrate, regulate albumen or co-factor in conjunction with or related any other zone in one or more amino acid mutations (for example, replacement).Aminoacid replacement comprises that at least one amino acid of replacement is up to the replacement entire domain or more than a territory (as several SCR); Or above combination.Except that replacing, also can carry out interpolation and disappearance to one or more amino acid residues or territory.

In some embodiments, with the member's of complement protein enzyme family protease domain with (a) complement family second the member's or (b) member's of serine protease superfamily protease domain replace.(a) example is the protease domain that replaces factor I with the protease domain of factor B.(b) example is the replacement with the protease domain of chymotrypsin or elastoser.These replace the substrate specificity that can change described complement proteases.For example, thus the substrate specificity that can change C3 convertase makes C3 convertase can cut toxin rather than its normal substrate.In some variant, analog can significantly lack proteinase activity or have the proteinase activity of reduction but can for example combine with detectable binding affinity with complement component.

Discuss as this paper, the present invention provides in addition with the method that suppresses complement activity as complement protein analogs such as factor B analogs.As shown in hereinafter some embodiment, the natural factor B from species may have activity in the complementary reaction/approach from another species in some cases.Therefore, the present invention also comprises the complement protein analog from species, is used for suppressing the complement activity of another species.

In some embodiments, the complement protein analog can comprise with naturally occurring complement component on the different glycosylation pattern of glycosylation pattern, perhaps lack glycosylation fully.Be used for for comprising that N-connects glycosylation or O-connects glycosylated glycosylation site polypeptide of sequence analog, can be such as (for example using dog pancreatic microsome system, see Mueckler and Lodish (1986) Cell 44:629 and Walter, P. (1983) Meth.Enzymol.96:84) wait and add in this polypeptide analog or from wherein remove external sugar.The production process of complement protein analog of the present invention can comprise add or disappearance/sudden change corresponding to the amino acid sequence of glycosylation site, for example, the glycosylation pattern/state of change albumen can change the functional characteristic of albumen.For example, fb2 and fb3 (factor B analog) comprise the N285D replacement of removing the N-glycosylation site.The forfeiture of described N-glycosylation site has changed the feature of described albumen.By at glycosylation pattern or this N285 is not carried out in the glycosylated cells producing described albumen and can reach identical effect with change.For example, can N285 not carried out producing in the glycosylated Bacillus coli cells fB albumen or analog.In some embodiments, except 285 amino acids of correspondence were asparagine, the albumen that comprises the sequence of fb2 or fb3 can produce in the glycosylated cells (for example, Bacillus coli cells) not carrying out 285 amino acids.

In some embodiments, composition of the present invention or molecule are carried out Pegylation (PEGylate), for example, see Roberts etc., Advanced Drug Delivery Reviews 54 (4): 459-476 (2002); Veronese, Biomaterials 22 (5): 405-417 (2001); Fee and Alstine, Chemical Engineering Science 61 (3): 924-939 (2006); Kodera etc., Progress in Polymer Science 23 (7): 1233-1271 (1998); Morar, BiopharmInternational 19 (4): 34 (2006); And Veronese and Pasut, Drug Discovery Today10 (21): 1451-1458 (2005).

In some embodiments, c-terminus and/or aminoterminal have been carried out chemical modification.Can in various embodiments of the present invention, introduce as acidylate (for example, acetylization) or alkylation aminoterminals such as (for example, methylating) and modify and as c-terminuses such as amidatioon modification and comprise that other of cyclisation is end modified.To some aminoterminal of core sequence and/or c-terminus is modified and/or peptide prolongs (for example with as the fusion of albumin, immunoglobulin or its part heterologous polypeptide such as (as the immunoglobulin Fc territories)) favourable physics, chemistry, biochemistry and pharmacological property can be provided, for example: the stability of enhancing, raising tire and/or usefulness, to the opposing of haemocyanin enzyme and required pharmacokinetic property or the like.

The present invention and then analog as complement protein fragment (fragment that comprises the complement protein analog) is provided, described complement protein fragment contain at least 10,20,50,100,200,300,500 of target proteins or 600 amino acid whose parts and/orcomprise 1,2 of described albumen or 3 territories and have wild type complement protein or analog more than one biologically active and/or serve as the inhibitor of an aspect (classical pathway and/or alternative route) of complement system.

Various technology that can be by including but not limited to chemosynthesis or the analog by express recombinant prepare analog of the present invention.

For example, factor B can be conditioned the example of the target molecules of (for example, suppressing) as activity.In addition, for example, this paper has described the particular analog of factor B.Can operate factor B with multiple formula, thereby for example suppress or reduce the activation of alternative route.For example, factor B binding molecule (molecule of deriving as antibody or fit) can in conjunction with and/or isolation factors B, thereby for example described molecule can not help the formation of invertase.Can with as inhibition molecule local expressions such as RNAi molecule, ribozyme or catalytic antibodies so that prevent the expression of factor B or with its destruction.In some embodiments, can be for example change specific site in the factor B, thereby described molecule is brought into play function no longer entirely truely by rite-directed mutagenesis.In some embodiments, for example by becoming another kind of amino acid (as N, A, E, S, Y or G) from D corresponding to the residue of 740 amino acids of people's factor B (shown in SEQ ID NO:2), can change the enzyme part or the enzyme territory (protease of described molecule, this protease is serine protease) thus make described molecule no longer have enzymic activity or have the enzymic activity (for example, having reduced at least 2 times, 5 times, 10 times, 50 times or 100 times) of reduction.The amino acid whose numbering of specificity factor B relates to the whole polypeptide that comprises signal peptide and is reflected among the SEQ ID NO:2 herein.Can reformed other site in the factor B comprise: 1) properdin binding site (properdin is in conjunction with the territory) thus carry out combination with lower affinity (for example, as compare the affinity that has reduced by 2 times, 5 times, 10 times, 50 times or 100 times with wild type factor B) or higher affinity (as compare the affinity that has increased by 2 times, 5 times, 10 times, 50 times or 100 times with wild type factor B); 2) C3b binding site (C3b is in conjunction with the territory) thus (reduced by 2 times with lower affinity as comparing with wild type factor B, 5 times, 10 times, the affinity of 50 times or 100 times) or higher affinity (increased by 2 times as comparing with wild type factor B, 5 times, 10 times, the affinity of 50 times or 100 times, for example, this can be by realizing corresponding to 279 of SEQ ID NO:2 and/or 285 amino acid with other aminoacid replacement, for example, wherein use G, A or N replace corresponding to the amino acid of 279 bit positions and/or with D or A and replace amino acid corresponding to 285 bit positions) carry out combination; 3) site of factor D role, thereby make factor D have the cutting power of reduction or no longer cut factor B (for example to form Bb, at factor D cleavage site place, at least one amino acid corresponding to 258,259 or 260 bit positions of SEQ ID NO:2 can be become for example A); Perhaps above-mentioned 1,2 and/or 3 combination.Because factor B has central role in complement activation, thereby factor B is attractive target molecules.

About 200 amino acid of " vWF ELISA (vWF) " territory (also claiming A type territory) average out to.It occurs invWF 3 times and once (Columbatti and Bonaldo (1991) Blood 77:2305 summary) occur in factor B, C2, CR3 (Mac-1), CR4 and other albumen.Complete sequence similitude between the vWF territory is generally 18%～64%.

The present invention also provides and has demonstrated increase, reduced people's factor B of modification of the lysis activity of the complement-mediated maybe can't detect.

The factor B analog of some modification of the present invention comprises more than one amino acid change as discussed herein and additionally has more than one additional amino acid and replaces, inserts or (for example change, at least or be no more than 1,2,5,8,10,15,20,50,100 or 200 change), described analog has kept and the combining or other biologically active of factor B analog as discussed herein of the increase of C3b and/or factor D, thereby mediation is to the inhibition of complement pathway.These analogs can have at least 99.9%, 99%, 98%, 95%, 90%, 85%, 80%, 75% or 70% and amino acid sequence identity wild type factor B (for example, the amino acid sequence of SEQ ID NO:2) and keep combination to the increase of C3b and/or factor D.

With wild type factor B the combination of C3b and/or factor D is compared, some factor B analog of the present invention can have has increased by 2 times, 4 times, 5 times, 10 times, 20 times, 50 times, 100 times, 500 times, 1000 times the combination to C3b and/or factor D.

Some embodiment of the present invention is led to relate to and be can be used for producing modified complement pathway composition (for example, polynucleotides fB3) and host cell (or many cells host living beings).Complement pathway composition that separates these modifications and the method for testing the activity of its complement-mediated also are provided.Some aspect of the present invention relates to such pharmaceutical composition: the complement pathway composition of wherein said modification is at the active component that treats and/or prevents under the environment.Some embodiment of the present invention also relates to the method with the complement protein treatment complement-mediated illness of the modification of treatment effective dose.

Some embodiment of the present invention has adopted the binding molecule that can combine with target molecules (for example, antibody or its fragment).In some embodiments, binding molecule suppresses as participates in the activity of the target molecules such as molecule of complement relational approach.

In some embodiments, adopted the binding molecule that can combine, for example, seen that No. the 20050260198th, U.S. Patent application and PCT disclose WO0021559 number with factor B and/or factor D.In some embodiments, binding molecule combines and is combined in factor B in the 3rd short total duplicate domain.

Some embodiment of the present invention can adopt peptide, albumen or other molecule (for example, antibody or fit) that can combine with the composition of complement relational approach.In some embodiments, peptide or other molecule and a kind of composition combine and block another kind of composition combination or with its competition.In some embodiments, peptide or other molecule and a kind of composition combine and block or suppress the activity (for example, the catalytic domain of blocking-up enzyme) and/or the blocking-up of described composition can be by for example site of another kind of composition effect.In these embodiments, the site that can be applied can be for cleavage site or as another site (for example, phosphorylation site or dephosphorylation site) of the substrate of the enzyme activity.In some embodiments, used peptide simulated factor B in conjunction with character but lack to activate the ability of C3, for example, see that PCT discloses WO0021559 number.In some embodiments, can use peptide as herein described or albumen, perhaps can express them with nucleic acid or carrier.In some embodiments, a plurality of peptides have been adopted to a plurality of sites and/or composition effect.

The present invention includes the molecule that (i) combines with factor C3b and factor D simultaneously, for example, bispecific antibody fB3 etc.; (ii) (compare) the complement protein analog that has the adhesion of factor C3b and factor D increase with its native form; (iii) (compare) the complement protein analog that has the adhesion that factor D is increased with its native form; (iv) (compare) the complement protein analog that has the adhesion that the C3bB compound is increased with its native form.The present invention also comprises the method for using molecule of the present invention (as the molecule of above-mentioned i～iv) to suppress complement pathway.In some embodiments, the molecule that combines with factor C3b and factor D while is not wild type fB.In some embodiments, the molecule that combines with factor C3b and factor D while is not fB1, fB2 or fB3.

In some embodiments, the increase multiple of the combination that increases is about 1.5～about 10,000, about 10～about 10,000, about 100～about 10,000, about 1,000～about 10,000, about 1.5～about 1,000, about 1.5～about 100, about 1.5～about 10, about 2～about 5, about 2～about 10, about 5～about 10, about 5～about 20, about 10～about 20, about 10～about 30, about 20～about 30, about 30～about 50, about 50～about 100, about 100～about 500, about 500～about 1,000, about 1,000～about 5,000 or about 5,000～about 10,000.In some embodiments, the increase multiple of the combination of increase is greater than 1.5,2,3,4,5,10,50,100,500,1000,5000 or 10,000.In some embodiments, can for example relatively measure combining of increase by immunoprecipitation with wild-type protein.For example for above-mentioned (i), can by use to the binding molecule of C3b to this albumen carry out immunoprecipitation then for example with as immunoassays such as ELISA or Western detect in the immunoprecipitate D (for example, in conjunction with increase shows as the band that intensity increases among the Western) thus the mensuration combination.

In some embodiments, epi-position in the Three S's CR territory of binding molecule and factor B combines, and described epi-position is selected from: (a) comprise the epi-position corresponding to the factor B of about 164 that comprise SEQ ID NO:2～about 210 at least a portion people's factor B or the position that is equal to it in non-human factor B sequence; (b) comprise corresponding at least a portion people's factor B of about 164 that comprise SEQ ID NO:2～about 166 amino acids sequences or in non-human factor B sequence with the epi-position of the factor B of its equivalent site; (c) comprise corresponding at least a portion people's factor B of about 207 that comprise SEQ ID NO:2～about 210 amino acids sequences or in non-human factor B sequence with the epi-position of the factor B of its equivalent site; (d) comprise corresponding to comprise more than any one the amino acid whose at least a portion people's factor B of following column position or in non-human factor B sequence with the epi-position of the factor B of its equivalent site: 164,165,166,207,209 or 210 of SEQ ID NO:2.Aspect another, epi-position in the Three S's CR territory of antibody or its Fab and factor B optionally combines, and described epi-position comprises the amino acid corresponding to more than one following amino acid position or its equivalent site in non-human factor B sequence: 162,164,166,207,210,214,215 and 217 of SEQ IDNO:2.On the other hand, epi-position in the Three S's CR territory of described antibody or its Fab and factor B optionally combines, and described epi-position comprises the amino acid corresponding to following amino acid position or its equivalent site in non-human factor B sequence: 162,164,166,207,210,214,215 and 217 of SEQ ID NO:2.Aspect another, epi-position in the Three S's CR territory of described antibody or its Fab and factor B optionally combines, and described epi-position is by forming corresponding to the amino acid of following amino acid position or its equivalent site in non-human factor B sequence: 162,164,166,207,210,214,215 and 217 of SEQ ID NO:2.Described antibody or Fab can combine with the non-linear epi-position in the three-dimensional structure in the part Three S's CR territory of factor B, and wherein said part is limited by the amino acid position corresponding to the equivalent site in 162～217 of SEQ ID NO:2 or the non-human factor B sequence at least.

In some embodiments, binding molecule combines with factor B and suppresses, prevents, reduces and/or eliminate and (ablate) formation of C3bBb compound.In some embodiments, binding molecule combines with factor B and has suppressed, prevents, reduced and/or eliminated the cutting of factor D to factor B.In some embodiments, binding molecule competitiveness suppressed monoclone antibody 1379 (produce by hybridoma with ATCC storage PTA-6230, American Type Culture Collection, P.O.Box 1549, Manassas, VA 20108) with the combining of people's factor B.In some embodiments, binding molecule is monoclone antibody 1379 (ATCC storage PTA-6230), the humanization form of monoclone antibody 1379 or chimeric form, perhaps any Fab of antibody 1379 or its humanization form, for example: the CDR1 that comprises the heavy chain variable domain of antibody 1379, the CDR1 in CDR2 and CDR3 and/or light chain variable territory, CDR2 and CDR3 (can lack in case of necessity, insert and/or replace among the describedCDR 1,2,3,5,10,12,15 or 20 amino acid are to improve binding affinity or other kinetic parameter) antibody; Or by for example ELISA or the definite antibody that combines with monoclone antibody 1379 competitions of other immunoassay (preferred humanized antibody or human antibodies).

In some embodiments of the present invention, regulated the activity of (for example, suppressing) factor D.In some embodiments, this carries out with the adjusting combination to the activity (for example, the activity of the activity of the activity of factor B, C3b and/or C3bB) of another kind of pathway components.The serum-concentration that it is believed that factor D is relatively low.In some embodiments, factor D can be that for example antibody, inhibition analog or soluble recepter fit, pathway components are isolated tactful target molecules.Can obtain factor D " inhibition " molecule such as in it and the local expression in antibody or the fit space between RPE cell and Bruch's membrane by practice of the present invention.In some embodiments, can use as factor D such as albumen " inhibition " molecule (for example, by to ocular injection).For example, United States Patent (USP) the 6th, 956 relates to the factor D inhibitor No. 107.In some embodiments, antibody or its factor D binding fragment have been adopted, as monoclone antibody 166-32 (accession number HB-12476, ATCC), any Fab of the humanization form of monoclone antibody 166-32 or chimeric form or antibody 166-32 or its humanization form, for example: the CDR1 that comprises the heavy chain variable domain of antibody 166-32, the CDR1 in CDR2 and CDR3 and/or light chain variable territory, CDR2 and CDR3 (can lack in case of necessity, insert and/or replace and have 1 among the described CDR, 2,3,5,10,12, thereby 15 or 20 amino acid improve binding affinity or other kinetic parameter) antibody; Or by for example ELISA or the definite antibody that combines with monoclone antibody 166-32 competition of other immunoassay (preferred humanized antibody or human antibodies).

Another composition of alternative route is CFH.In some embodiments of the present invention, regulated the activity of (for example, suppressing) CFH.In some embodiments, this carries out with the adjusting combination to the activity (for example, the activity of the activity of the activity of factor B, C3b and/or C3bB) of another kind of pathway components.For example, 402 the tyrosine of CFH is relevant with low AMD risk, yet relevant with excessive risk at the histidine of this position.The Y402H polymorphism that is arranged in the SCR7 of CFH also sees CFH splicing variants FHL-1 (Estaller etc., 1991; Sim etc., 1993).CFH shows similar complement regulatory function (Zipfel ﹠amp with FHL-1; Sherka, 1999).The CFH performance prevents the function of the important inhibitor of not controlled complement activation.Tyr402his polymorphism through identifying might be relevant with inflammation of eye section.Thereby the therapeutic code area can be the nucleic acid of 402 tyrosine of coding CFH or FHL-1 molecule.Be converted to carry and express the cell of described molecule and will be within the eye for example have favourable, result of treatment and/or preventive effect for inflammation.

When being appreciated that the nucleic acid of introducing encoding proteins when touching upon, the present invention has also considered to introduce described albumen itself.Be appreciated that the present invention has also considered the introducing nucleic acids encoding said proteins when touching upon introducing albumen.In some embodiments, albumen and its nucleic acid of coding have been introduced simultaneously.

In addition, can be with nucleic acid of the present invention or albumen for example as cell therapy via cell delivery or be administered in the animal.For example, if use or carry specific protein, this can be by using or carrying the cell of expressing described albumen to finish.In some embodiments, come from cellular expression albumen by promotor adjustable, derivable and/or that can prevent.In some embodiments, with expressing being transported in the animal of protein of interest and/or nucleic acid, for example, see that PCT discloses WO07078922 number through encapsulation of cells.The cell that animal is used can be autogenous cell, homogeneous variant cell or heterogenous cell.In some embodiments, thus to autogenous cell take out external handle to make them produce molecule of the present invention and then described cell introduced get back to described animal.In some embodiments, with cell local application (for example, in the joint, in the vitreum, in the retina or the like) or systemic application (for example, intravenous injection).

In some embodiments, the target cell is as mammalian cells such as primate cell and human cells.In some embodiments, the target cell is as eye cells such as RPE cell, retina cell or pluripotent cells.The target cell can be for cell in vitro, take from cell or cells in vivo in the body.In some embodiments, the gene delivery system of this paper consideration can cause interested gene or the code area stable integration in the host cell gene group.In some embodiments, cell is a stem cell.Stem cell includes but not limited to multipotential stem cell, myeloid-lymphoid stem cell, candidate stem cell, tumor stem cell and embryonic stem cell.In some embodiments, the pluripotent cell of this paper consideration is not to be used for breeding those pluripotent cells that living individual is arranged from fertilized egg or blastomere.The present invention has also considered the part neoblast is used for implantation at the patient's of needs treatments intraocular for example so that make a cytothesis.

The example of interested gene or therapeutic gene includes but not limited to the nucleic acid of encoding as the expression product of the expression of inflammation inhibitors such as DAF to strengthening, and described inflammation inhibitor can reduce the expression of inflammation-induced agent or promoter or the like.

In some embodiments, molecule of the present invention is fit.Fit nucleotide sequence similar with antibody, that combine with target molecules.This technology provides the nucleic acid molecules that has unique sequences separately, described nucleic acid molecules has the character that combines with required target compound or molecular specificity, but this may not be the complementary base pairing by a chain and another chain, but is similar to part and its acceptor or companion's the reaction or the reaction of antigen and antibody.Nucleic acid molecules can be the ligands specific of given target compound or molecule.Thereby nucleic acid has the enough abilities that form multiple 2 and 3 dimensional organization and has enough chemical versatilities and serves as no matter be the part of almost any compound of monomer or polymer or combination (form specificity in conjunction with to).The molecule of any size can both for example be seen as target, United States Patent (USP) the 5th, 567, No. 588, the 5th, 270, No. 163 and the 5th, 756, No. 291.Can be similar to antibody as herein described or binding molecule described such use fit.In some embodiments, of the present invention fit be sulfo-fit (thioaptamer for example, sees Volk etc., Annals of the New York Academy of Sciences 1082 (1), 116-119 (2006)).

In some embodiments, molecule of the present invention or binding molecule are as ADNECTIN^TMDeng antibody analog, also for example see United States Patent (USP) the 7th, 115, No. 396.ADNECTINS^TMBe the target biology goods that a class is derived from fibronectin.

Usually, be used to identify that fit external selection technology relates to has the dna molecular storehouse that contains some random areas or mutagenesis zone at least.Thereby the oligonucleotide library that is used for fit selection can contain the zone of 20～100 random nucleotides, and the side in described zone for example can be useful in conjunction with PCR primer about 15～25 base districts of sequencing row really.Can be with the standard round pcr described oligonucleotide library that increases, however any method of the amplification that nucleotide sequence can be provided also can be adopted.Then thereby described storehouse is produced the rna transcription thing in in-vitro transcription.Then thereby described rna transcription thing execution selection scheme being isolated with another molecule is the nucleic acid of part (for example, albumen or any target molecules) specificity combination.Can carry out reverse transcription and amplification to the RNA molecule that combines with described part then.Can carry out extra selection step to the sequence of choosing.Thereby can increase, clone its cDNA then and the candidate that further characterizes described target part of checking order fit.In case successfully determined fit sequence, thus just can with described fit further optimize or modify by extra selection step by mutagenesis obtain for example higher molecule of specificity.What advantageously, selection combined with part in the presence of the salinity of simulating normal physiological conditions and temperature is fit.

The same with many nucleic acid used herein, interested nucleic acid can be DNA or RNA, and can be single chain molecule, perhaps can be the molecule of partially double stranded or complete two strands.In some embodiments, nucleic acid can be but be not limited to DNA, RNA, miRNA or siRNA.In some embodiments, nucleic acid is encoded to albumen.In some embodiments, nucleic acid is not encoded to albumen.In some embodiments, nucleic acid suppresses from second nucleic acid (for example, protein expression mRNA).

The same with any therapeutic nucleic acids, fit can the combination with target proteins, or can combine in the code area or in as noncoding regions such as intron or upstream/downstream adjusting sequences no matter be with target nucleic acids.

There are a lot of modes can modification of nucleic acids not weaken the nucleic acid of described nucleic acid of expectation in conjunction with character so that obtain relative favourable character.These modes are known for those skilled in the art and comprise Pegylation (PEGylation), sulphur backbone modifications and methylate.

" nucleic acid molecules of stabilisation " should refer to relatively to resist the nucleic acid molecules of vivo degradation (for example, via exonuclease or endonuclease).Stabilisation can be the function of length and/or secondary structure.Can by control example if can stable molecule secondary structure obtain stabilisation.For example, if 3 ' end and upstream region complementation of nucleic acid molecules, then this part can be folded back and form " the stem ring " of stablizing described molecule.

" ribozyme " is meant the nucleic acid that can cut specific nucleic acid sequence.In some embodiment, ribozyme should be interpreted as to refer to the RNA molecule that contains the antisense sequences that is useful on specific recognition and RNA cutting the enzyme activity, for example see United States Patent (USP) the 6th, 770, No. 633.

Thereby antisense oligonucleotides normally influences the small oligonucleotide of this expression of gene with the part complementation of gene.Can come inhibition of gene expression by the hybridization of oligonucleotides and specific gene or its mRNA (mRNA).In some cases, can the application of treatment strategy weaken it is believed that and to start or to quicken a kind of of inflammation or several expression of gene, for example see United States Patent (USP) the 6th, 822, No. 087 and WO 2006/062716.

" siRNA " or " short interfering rna " or " siRNA " or " short hairpin RNA " or " shRNA " are that RNA disturbs the form of (RNAi).RNA interfering can be and target nucleic acids sequence (for example, VEGF) complementary double stranded rna molecule or partially double stranded RNA molecule.Little RNA interfering (miRNA) also belongs to this classification.Double stranded rna molecule forms by the complementary pairing of described intramolecular RNA part with the 2nd RNA part.The length of each several part is usually less than 30 nucleotide long (for example, 29,28,27,26,25,24,23,22,21,20,19,18,17,16,15,14,13,12,11 or 10 nucleotide).In some embodiments, the length of each several part is that 19～25 nucleotide are long.In some siRNA molecule, the first of the complementation of described RNA molecule and second portion are hairpin structure " stems ".Described two parts can be connected by catenation sequence, and described catenation sequence can form " ring " in the described hairpin structure.The length of described catenation sequence can be different.In some embodiments, described catenation sequence can be long for 5,6,7,8,9,10,11,12 or 13 nucleotide.Catenation sequence can be used for connecting described first and second portion, and is known in the art.Complementary but the incomplete symmetry of described first and second portion, this is to give prominence to nucleotide (for example, 1,2,3,4 or 5 nucleotide is outstanding) because described hairpin structure can comprise 3 ' or 5 '.RNA molecule of the present invention can be from vector expression or by chemistry or synthetic the generation.

MiRNA is by regulating the little RNA that posttranscriptional gene is expressed with the interaction of homologous mRNA.MiRNA can be by expressing with the controlling gene that combines from the complementary site of the target mRNA of protein coding gene.Can handle from bigger double-stranded precursor molecule and obtain miRNA.Described precursor molecule normally is about the hairpin structure of 70 nucleotide, and 25 above nucleotide base pairings in this hair clip are arranged.Thereby RNA enzyme III sample enzyme Drosha and Dicer cut described precursor and produce miRNA.MiRNA normally strand and incorporating into is called in the albumen composition that RNA induces reticent compound (RISC) or miRNP.The mRNA of RNA albumen composition target complementation.The translation of miRNA inhibition target mRNA or guidance are to cutting (Brennecke etc., the Genome Biology 4:228 (2003) of target mRNA; Kim etc., Mol.Cells 19:1-15 (2005)).The inhibition to expression of nucleic acid of RNAi mediation has relative specificity.Some base-pair mismatch between the nucleic acid of RNAi molecule and institute's target can be tolerated and can not abrogated the effect that RNA disturbs, for example be seen WO 2006/062716.RNAi of the present invention can not cause significant antiviral response usually.

The scheme that has known being used to design siRNA in this area (is for example seen Elbashire etc., 2001, Nature, 411:494-8; Amarzguioui etc., 2004, Biochem.Biophys.Res.Commun., 316 (4): 1050-8; With Reynolds etc., 2004, Nat.Biotech., 22 (3): 326-30).The details of preparation siRNA is found in the website as some commercial distribution merchants such as Ambion, Dharmacon, GenScript, Invitrogen and OligoEngine.Usually can compare program (seeing National Library of Medicine internet site) with BLAST and check that the sequence of any potential siRNA material standed for and any of polymorphism of other nucleotide sequence or nucleotide sequence may mate.Usually, produce and screen many siRNA, see United States Patent (USP) the 7th, 078, No. 196 so that obtain effective drug candidates.SiRNA of the present invention can be from vector expression and/or by chemistry or synthetic the generation.Synthetic RNAi can (Carlsbad, CA) etc. commercial source obtains from for example Invitrogen.The RNAi carrier also can obtain from commercial source such as for example Invitrogen.

Three chain molecules be meant unique DNA chain and target double-stranded DNA by combining major groove and forming conllinear three chains (co-linear triplex) thus prevent from or change to transcribe (for example seeing United States Patent (USP) the 5th, 176, No. 996).

Can express a plurality of molecules by recombination method in conjunction with target.For example, can use single-chain antibody, domain antibodies and acceptor etc., thereby and their nucleic acid of will encoding prepare in practice of the present invention as therapeutic gene and treats albumen.

" antibody " is meant complete immunoglobulin or its antigen-binding portion thereof, and described antigen-binding portion thereof combines specificity with complete antibody and is at war with, and promptly described fragment has kept the antigen binding capacity of homology.In some embodiments, can produce antigen-binding portion thereof by recombinant DNA technology or by enzyme cutting or chemical cleavage to complete antibody.In addition, antigen-binding portion thereof comprises F_Ab, F_{Ab '}, F_{(ab ') 2}, F_v, dAb, and complementary determining region (CDR) fragment, single-chain antibody (scF_v), chimeric antibody, bivalent antibody (diabody) and contain the polypeptide that is enough to polypeptide is given immunoglobulin at least a portion of specific antigen combination.F_AbFragment is by V_L, V_H, C_LAnd C_HThe unit price fragment that 1 territory is formed; F_{(ab ') 2}Fragment is two F that comprise by the disulfide bridge connects of hinge area_AbThe divalence fragment of fragment; F_dFragment is by V_HAnd C_H1 territory is formed; F_vFragment is by the V of the single armed of antibody_LTerritory and V_HThe territory is formed; DAb fragment (Ward etc., Nature 341:544-546,1989) is by V_HThe territory is formed.Single-chain antibody (scF_vOr scAb) be antibody derivatives, V wherein_LDistrict and V_HThereby the district forms monovalent molecule via synthetic linker pairing, thereby makes and described V district can be made single protein chain (Bird etc., Science 242:423-426,1988 and Huston etc., Proc.Natl.Acad.Sci.USA85:5879-5883,1988).Bivalent antibody is the bifunctional antibody of divalence, wherein V_LTerritory and V_HExpress on the single polypeptide chain in the territory, but used joint is too short described two territories are matched on same chain, thereby force the complementary territory pairing of described territory and another chain and produced two antigen binding sites and (for example see Holliger, P. etc., Proc.Natl.Acad.Sci.USA 90:6444-6448,1993, and Poljak, R.J. etc., Structure 2:1121-1123,1994).One or more CDR covalently or non-covalently can be added in the molecule.As known in the art, single CDR can give antigen binding capacity and specificity to the polypeptide that unloads it.The molecule of specificity combination is such molecule: identify and estimate the standard of specificity and cross reactivity from a plurality of other molecules identifications and in conjunction with the epi-position (isogeneic) that is used to produce original antibody thereby have in the necessary available field of immunology of concentrated reactivity known being used for.Can adopt any described antibody in the present invention or keep the described antibody fragment of some its adhesion at least.Be used to prepare, purifying, to use and/or utilize the whole bag of tricks of antibody be known in the art, for example sees United States Patent (USP) the 6th, 884, No. 879.

Chimeric antibody is the molecule that contains the different piece that is derived from the different animals species, as has the variable region that is derived from muroid mAb and those molecules of human immunoglobulin(HIg) constant region.The preparation of chimeric antibody and use are mainly in order to reduce immunogenicity.Chimeric antibody and production method thereof are (Morrison etc., Proc.Natl.Acad.Sci.USA 81:6851-6855 (1984) known in the art; Boulianne etc., Nature 312:643-646 (1984); European patent application 125023; Neuberger etc., Nature 314:268-270 (1985); European patent application 171496; European patent application 184187; Sahagan etc., J.Immunol.137:1066-1074 (1986); Liu etc., Proc.Natl.Acad.Sci.USA 84:3439-3443 (1987); Sun etc., Proc.Natl.Acad.Sci.USA 84:214-218 (1987); Better etc., Science 240:1041-1043 (1988); With Harlow ﹠amp; LaneAntibodies:a Laboratory Manual Cold Spring Harbor Laboratory (1988)).

Term used herein " Humanized immunoglobulin " is meant the immunoglobulin of the immunoglobulin part that comprises separate sources, and wherein at least one part is derived from the mankind.For example, the part that humanized antibody can comprise the immunoglobulin that has essential specific non-human source (for example, from mouse) and the part (for example gomphosis immunoglobulin) of the immunoglobulin sequences of human origin, described part by routine techniques (for example, synthetic technology) chemically connected together or is prepared as the polypeptide (for example, thereby the DNA that can express the protein part of the described chimeric antibody of coding produces the polypeptide chain of adjacency) of adjacency with technique for gene engineering.Another example of Humanized immunoglobulin of the present invention is the immunoglobulin that contains more than one immunoglobulin chain, described immunoglobulin chain comprise the CDR that is derived from non-human source antibody and be derived from the light chain of human origin and/or the framework region of heavy chain (for example, CDR grafted antibody, this antibody can have or not have in order to the framework that improves antibody stability and/or binding affinity change and can have in case of necessity in more than one CDR in order to improve changing more than of antibody stability and/or preferred combination affinity).The term Humanized immunoglobulin also comprises single-chain antibody chimeric or that CDR transplants, for example sees Cabilly etc., United States Patent (USP) the 4th, 816, No. 567; Boss etc., United States Patent (USP) the 4th, 816, No. 397; Neuberger etc., WO 86/01533; Winter, United States Patent (USP) the 5th, 225, No. 539; Padlan etc., european patent application the 0th, 519, No. 596, Ladner etc., United States Patent (USP) the 4th, 946, No. 778; Huston etc., United States Patent (USP) the 5th, 476, No. 786; Studnicka etc., United States Patent (USP) the 5th, 766, No. 886; Queen etc., United States Patent (USP) the 7th, 022, No. 500 and Bird etc., Science, 242:423-426 (1988).

Catalytic antibody is to influence the interior chemical reaction of combined antigen or the antibody of chemical change (as the cutting of peptide bond).Can be by animal immune being come inducing catalysis antibody (Pollack etc., J.Am.Chem.Soc. (1988) 110:8713, Jackson etc. with having given immunogenic transition state analog (TSA), PNAS (1988) 85:4953, Shokat etc., Chem.Int.Ed.Engl. (1988) 27:1172), and the dissimilar chemical reaction of catalytic antibody energy catalysis is for example seen United States Patent (USP) the 5th, 401, No. 641, the 6th, 590, No. 080, the 7th, 109, No. 291 and the 7th, 205, No. 136.In some embodiments, catalytic antibody cause target proteins go stablize.In some embodiments, catalytic antibody serves as and cuts for example protease of target molecules such as factor B, factor D, factor B b, factor C3 and/or factor C3b.

In addition, can use the antibody of the camellid of natural shortage light chain.Can use the structure that is called nano antibody and domain antibodies, contain the known polypeptide that has kept the single CDR of the antibody that antigen, determinant or epi-position binding ability just can combine with isogeneic as long as comprise.

The binding molecule that produces through reorganization of single-chain antibody (" SCA ") and other form can be used for for example gene conveying.The correlative coding sequence of specific antibodies and antigen-binding portion thereof thereof can be separated and clones to be used to prepare the polypeptide that has essential isogeneic binding ability.The method for preparing these fragments is known in the artly (for example to see Harlow ﹠amp; Lane, Antibodies:A LaboratoryManual, Cold Spring Harbor Laboratory, New York, 1988).

In some embodiments of the present invention, antibody sequence is humanized antibody sequence or human sequence antibody, for example in order to reduce the risk that it is produced immune response.Can take various steps known in the art to come the amino acid of modified antigen, thereby when making this molecular antigen littler, keep in conjunction with active in conjunction with polypeptide or interested any treatment albumen by the amino acid that replaces interested (determined) specific site place as design alternative.

In some embodiments, aminoacid replacement includes but not limited to: (1) reduces proteoclastic sensitivity, (2) reduction is to the sensitivity of oxidation, (3) change the binding affinity that is used to form albumen composition, (4) (for example change, increasing or reduce) binding affinity and (5) reduce immunogenic aminoacid replacement.

The present invention also comprises the application of analog.Analog can comprise the various mutains of the sequence except that naturally occurring peptide sequence.For example, can in naturally occurring sequence, (for example in the polypeptide portion outside the territory that forms intermolecular contact) carry out monamino acid replacement or amino acids replacement (for example, conserved amino acid replaces).Conserved amino acid replaces the architectural feature (for example, alternative amino acid should not be tending towards destroying the spiral that exists in the parental array, or disintegrates the secondary structure of other type that constitutes the parental array feature) that should not change parental array in fact.At Proteins, Structuresand Molecular Principles (Creighton, Ed., W.H.Freeman and Company, NewYork (1984)); Introduction to Protein Structure (C.Branden and J.Tooze, eds., Garland Publishing, New York, N.Y. (1991)); With the art-recognized polypeptide secondary structure and the example of tertiary structure have been described among the Thornton et at.Nature354:105 (1991).In some embodiments, adopted non-conservative replacement.

In the situation of gene therapy, change will be reflected to the level of code nucleic acid.Thereby, also can modify so that strengthen expression described nucleic acid.For example, particular host cell may some codon of preference.Thereby, coding (recoding) again may take place in the place of some codon of preference (for example, mammalian expression systems and cell).The coding again of nucleic acid is the available design alternatives of those of skill in the art.

The method that can in practice of the present invention, use control to transcribe and/or translate.In some embodiments, these methods can submit to local loop because of carrying, and for example carry to the gene of eye cell.For example, can use the polypeptide of peptide-nucleic acid oligomer (PNA) (Eglon etc., Nature 365,566,1993) or bind nucleic acid, refer to the transcription factor that combines with any genomic dna sequence as being designed by the zinc of introducing through engineering approaches.These through engineering approaches transcription factors can raise or reduce any endogenous gene through design.

The whole bag of tricks that is used for gene therapy and gene transfer is known and any of these method can both be used to put into practice the present invention.Generality summary to gene transfer method comprises Goldspiel etc., Clin.Pharm.12:488-505 (1993); Wu ﹠amp; Wu; Biotherapy 3:87-95 (1991); Tolstoshev, Ann.Rev.Pharmacol.Toxicol.32:573-596 (1993); Mulligan, Science 260:926-932 (1993); Morgan ﹠amp; Anderson, Ann.Rev.Biochem.62:191-217 (1993); And May, TIBTECH 11 (5): 155-215 (1993).The method of recombinant DNA technology is known and can writes Current Protocols inMolecular Biology, John Wiley ﹠amp with reference to Ausubel etc.; Sons, NY (1993); And Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In one aspect, expression vector or vector construction body coding is as antibody derivatives such as single-chain antibodies.In some embodiments, interested expression vector expressing antibodies derivative in the human eye cell.

Some embodiment of this aspect relates to the conveying of vector construction body to animal (for example, people).Carrier or even albumen can be direct or indirect to people's conveying, in the situation of directly carrying, as (for example passing through injection, as in vitreum or subretinal injection in eye) etc. make people directly carrier or albumen as described in the contact, in the situation of indirectly carrying, at first external then will be in cell transformed be transplanted to patient's body with described carrier transformant.In some embodiments, these are autogenous cells through cell transformed.In some embodiments, these are homogeneous variant cells through cell transformed.Can be by any method (as the transfection of the gene transfer of the gene transfer of electroporation, microinjection, cytomixis, chromosome mediation, microcell mediation, spheroplast fusion, lipofection, microparticle bombardment, calcium phosphate mediation, the virus infections etc.) nucleic acid that will comprise the code area just transfer to and be organized in the cultured cells.In case of necessity, alternative mark can also be introduced in the cell.If adopt alternative mark, then cell can be placed and select down, so that for example enhancing is expressed and/or separated those cells that can express the code area of being shifted and (for example see Loeffler ﹠amp; Behr, Meth.Enzymol.217:599-618 (1993); Cohen etc., Meth.Enzymol.217:618-644 (1993); And Cline, Pharmac.Ther.29:69-92 (1985)).

Can recombinant cell (for example, at external autogenous cell or homogeneous variant cell through transforming) be carried the patient by the whole bag of tricks known in the art.For example, can like that cell be sealed before using as known in the art.In some embodiments, when sealing, described cell is not an autogenous cell.In some embodiments, the haemocyte (for example, candidate stem cell and/or hemopoietic progenitor cell) of will recombinating is used through intravenous.In some embodiments, eye cell and/or pluripotent cell can be injected directly into intraocular.Required cell concentration depends on required effect, animal state etc., and can be determined by the those skilled in the art that put into practice means known in the art.

Can by standard clinical techniques determine as the composition that comprises albumen of the present invention, nucleic acid or carrier granular in treatment, suppress the effective dose in disease, approach or the illness for example relevant with blind property illness in eye with prevention.In addition, can adopt testing in vitro to help determine the optimal dose scope where necessary.The accurate dosage that adopts in the preparation also will depend on the seriousness of route of administration and described disease or illness.In some embodiments, this can decide according to practitioner's judgement and/or patient's individual instances.Can come extrapolated effective dose from the dose response curve that is derived from testing in vitro system or animal model test system.

Some molecule (s) of interest needn't be carried by gene transfer method, and can use by any medicament delivery method known in the art (as by injection, instillation or the like).Thereby molecule, Complement Regulatory Protein, dominant complement protein, complement binding peptide or the albumen of the present invention includes and make that complement factor for example or complement regulate that molecule for example weakens, neutralizes, reduces that it expresses etc. is fit, mirror image isomerism is fit (spiegelmer), anitibody type molecule or antibody being derived, peptide nucleic acid (PNA) or the like are also prepared it as known in the art like that.Thereby, the molecule (s) of interest freeze-drying can be rebuild in use being used for, or it is existed with the liquid form that contains just like medicine acceptable diluent such as water, salt solution or buffer solutions, and described molecule (s) of interest can contain optional buffer solution, preservative, stabilizing agent etc.Preparation can be stablized under room temperature, desuperheat or refrigerator temperature, perhaps can it is freezing.Preparation also can be taked other form, as be made as tablet or be stored in capsule or storage agent (depot) in or the like.

Complement pathway and be used for the etiologic etiological disease model of human AMD

This section provides the background information of complement system and novel disease model so that help the understanding of the present invention.Three kinds of complement activation approach are arranged, classical pathway, alternative route andlectin pathway.Embodiment 8 and 9 has described the example of albumen of the present invention.In some embodiments, these albumen alternative route of complement activation that can weaken.Yet based on the mode that all three kinds of complement pathways intersect, these albumen can reduce by any caused inflammation in three kinds of complement pathways, thus its teiology to small part is related to complement activation disease treatment is provided.These diseases include but not limited to early stage AMD, moist AMD and ground pattern atrophy.

Complement pathway is an immune part that is called natural immune system, and the instant protection that provides defence to infect before body fluid and the cell-mediated immune system branch is being provided described natural immune system.These approach are made up of about 35 kinds of factors.They are by showing the dynamic (dynamical) cascade reaction of high-order and be activated and inactivation but be subjected to very good adjusting.Particularly, alternative complement pathway has been evolved to circulating fast by positive feedback loop.The inventor does not find any to the not only effective but also safe therapeutic complement inhibitor of having developed of treatment chronic disease, and this may be because the lasting systematicness blocking-up or the inhibition of complement activity may make the patient easily infected.

Albumen of the present invention and/or the carrier of expressing them advantageously can be used for systemic application in mammal and/or treatment chronic disease.For example, suppress complement pathway as complement protein analogs such as factor B analog of the present invention or factor D analogs by competing with combining of native protein.This makes and can weaken complement activity rather than block described approach fully.Therefore, complement activity might be adjusted downward to curative level (for example, alleviating some symptom or its seriousness) and not exclusively block complement activity.Thereby, avoid or reduced and blocking-up complement activity relevant risk, as the infection risk of increase.Therefore, the invention provides the method for the treatment of complement relevant disease (for example, chronic disease) by systemic application (in for example, the intravenous injection, peritonaeum or oral) albumen of the present invention or composition.

The activity that is not the former complement-mediated of specificity sensing challenge infection may cause significant attached injury to normal cell.Self avoid this attached injury, the several components of complement system to be designed specifically restricting normal cell around complement activity and the protection in order to make.These complement inhibitors see in the liquid phase (blood plasma) or as the conformity membrane albumen on the normal cell usually.A kind of concrete complement factor CFH is the main inhibitor of alternative route, and CFH circulates in blood plasma with high level usually but can combine with cell membrane, intercellular matrix composition and some plasma protein.

Figure 11 and 12 summaries have been described complement pathway, notice that they intersect at the C3b place.

Table 1 has been summarized some instrumentality of complement activation.

The instrumentality of table 1. complement activation

Albumen	Approach	Function
			??C1inh	Classical pathway	Blocking-up starts
??C4bp	Classical pathway	Decay is quickened and the CFI co-factor
			??DAF	Classics and alternative route	The acceleration that decays of C3 convertase
??MCP	Classics and alternative route	CFI co-factor (for liquid phase C3b)
			??CR1	Classics and alternative route	Decay is quickened and the CFI co-factor
??CFH	Alternative route	Decay acceleration, CFI co-factor and MAC inhibitor
			??CFI	Classics and alternative route	Degraded C3b and C4b
??CD59	Classics and alternative route	Suppress the MAC assembling
			Acrasin	Classics and alternative route	Suppress the MAC assembling
S albumen	Classics and alternative route	Suppress the MAC assembling
			??HRF	Classics and alternative route	Suppress the MAC assembling

The distribution of various inhibitor is relevant with its function.For example, DAF, MCP, CD59 etc. are the conformity membrane albumen on the host cell.On the other hand, CFH is in the liquid phase but can combines with some solid structure.

The etiologic etiological model of a kind of AMD is described

Although the present invention is intended to be subjected to any specific mechanism model to limit, the inventor infers the main teiology that can describe most of AMD situations with drag.Many factors have promoted the development and the course of disease of individual interior AMD.Described to quicken one group of factor of the described disease course in the Most patients for example with drag.And these factors can cause the early stage especially or quick course of disease in this etiologic etiological individuality easily suffering from genotype for a variety of reasons.

Early stage AMD is characterised in that at the RPE cell and is called gather (Fig. 1) of drusen between the lower membrane of Bruch's membrane.Drusen contains many compositions, and these compositions comprise some complement factor and the plasma protein that is called c reactive protein (CRP), and wherein CRP is regarded as the label of inflammation usually.CRP is the pentamer albumen that combines with various lipids and nuclear composition.CRP also plays following effect: 1) with phagocyte on the Fc receptors bind; 2) the early stage step in the activation CCP; 3) membrane-bound complement inhibitor DAF, MCP and CD59 are raised; With 4) combine with CFH and (for example see Johnson etc., 2006; Black etc., 2004; Mold etc., 1999; With Li etc., 2004).

Be not wishing to be bound by theory, the inventor infers, in early days among the AMD along with the gathering of drusen, CRP combines and is fixed on wherein with drusen.The CRP that is fixed then will play and attract phagocyte and stimulate them to swallow and remove the effect of drusen.CRP realizes above-mentioned effect by the ability of itself and Fc receptors bind and by producing bioactive fragment C4a and C3a.Yet the activation that produces the classical pathway of these fragments also produces C3b, and C3b can enter alternative route and circulate by positive feedback loop.Thereby unless alternative route is controlled, otherwise drusen will become the bank of the inflammation of increase.Although CRP raises DAF, MCP and CD59, DAF, MCP and CD59 are cell surface protein and the alternative route that possibly can't effectively control drusen inside.The inventor infers, for the uncontrollable activation (runawayactivation) that prevents alternative route and remarkable inflammation, CRP can be in conjunction with the CFH in the fixing glass wart also.According to this non-limiting model, if if CFH can not be effectively in conjunction with CRP or it alternative route that just can't effectively weaken, then remarkable inflammation may appear under retina.This may cause the tissue damage that increases along with the increase of the combination of CRP.Thereby whole process can circulate and finally cause comprising the carrying out property AMD of two courses of disease in latter stage (moist AMD and ground pattern atrophy (GA)).Can determine by extra acquired factor and inherent cause whether the patient develops and moist AMD and GA, described factor comprise the balance of short angiogenesis factor and anti-angiogenesis and for example stromatin enzyme and TGF-β instrumentality HtrA1 expression (for example, see Oka etc., 2004; Yang etc., 2006; With DeWan etc., 2006).

In this model, CRP is the part to the natural reset procedure of eyeground chip.Yet this does the time spent in execution, and CRP can evoke tissue damage by the activation that substitutes complement pathway potentially.Invalid control to alternative route throughout the year may cause carrying out property AMD.The invention provides the various embodiments that suppress alternative route.

The design of albumen of complement activation can weaken

For example the present invention includes can as albumen and/or via gene transfer (carrier that for example, comprises the code area of gene and/or encoding said proteins) thus carry the albumen of the alternative route of reduction complement activation.These albumen can overcome the obstacle of the development that hinders complement inhibitor (for example, being used for the complement inhibitor of illness in eye), for example comprise: 1) avoid long-term systemic immunosupress; 2) in blood otherwise the too high complement factor of inhibition obtains usefulness aspect horizontal; 3) make treatment albumen near retina and Bruch's membrane, obtain enough effectively level and distribution; 4) obtain the interior treatment protein active of drusen; 5) enough duration of the therapeutic conveying of acquisition treatment chronic disease; 6) obtain usefulness and do not disturb CCP activity in the eyeground; With 7) avoid or weaken immune response to therapeutic agent (for example, local immunity reaction).

The inventor has determined that the positive feedback loop in the reduction alternative route is a kind of method with whole approach downward modulation.Can realize such reduction by function and/or the level of disturbing fB, fD or properdin.In some embodiments, can raise or normalization realizes the reduction of approach by making as the function of natural instrumentalities such as DAF, MCP, CR1 or CFH.In some embodiments, their extracellular domain is expressed and may can be penetrated drusen.

The inventor has determined that the appropriate method of reduction alternative route feedback loop is to disturb the function or the level of complement factor B (fB).Some embodiment of the present invention is with the dominant strategy fB function that is used to weaken.Below for example understand three kinds of concrete dominant fB parts that respectively carry particular feature.

Complement factor circulates as the inactive protein enzyme and brings into play its function via two step processes.At first, thus it combines with factor C3b and forms instantaneous compound.Secondly, thus it cut by fD and produce Bb and activate the fB serine protease.Then properdin has been stablized the C3bBb compound.

The coding people's factor B and the gene of other complement protein and the nucleotide sequence of code area and the amino acid sequence of these albumen are known in the art.For example, the gene of coding people's factor B and other complement protein is found in ncbi database accession number NG_000013.The amino acid sequence that the coded sequence of factor B is found in ncbi database accession number NM_001710 and the preceding former albumen of factor B is found in ncbi database accession number NP_001701 or P00751.Ncbi database accession number P00751 is a former protein sequence before people's factor B.Sequence from other animal species also is known in the art.By relatively, in mouse factor B sequence, (for example, see ncbi database accession number P04186), Three S's CR territory is positioned at 761 amino acid whose 160～217 of being somebody's turn to do preceding albumen, and ripe muroid factor B albumen is crossed over 23～761.Preceding 22 amino acid of mouse factor B are burst (SEQ ID NO:16).

Albumen is 764 amino acid whose albumen (SEQ ID NO:2) and has the signal peptide of crossing over 1～25 amino acids before people's factor B.The ripe chain of factor B is corresponding to 26～764.Three SCR districts of people's factor B are SCR1 (also claimSushi 1, cross over about 35～about 100), SCR2 (also claimSushi 2, cross over about 101～about 160) and SCR3 (also claimSushi 3, cross over about 163～about 220).

First dominant body is called fB1 in three kinds of dominant bodies, and fB1 has changed an amino acid in the fB protease site.This fB part combines with C3b with normal affinity and dynamics, but when stablizing in the effect that is subjected to fD and by properdin, it does not play protease and does not form C3 convertase, for example replaces corresponding to 740 the amino acid of SEQ ID NO:2 (for example, D740N) with N.

Second the dominant structural reform that is called fB2 become the identical amino acid that is changed with fB1, but changed in addition C3b in conjunction with two additional amino acids in the territory (replacing 279 and 285 amino acid) corresponding to SEQ IDNO:2 in case increase fB2 to the binding affinity of C3b, for example, D279G, N285D and D740N change.The N-glycosylation site of a supposition is removed in the N285D replacement.

The third dominant body that is called fB3 merged fB2 increase C3b combination sudden change and knock out the sudden change of the binding site that is subjected to the factor D cutting, particularly corresponding to the replacement of the position of 258,259 and 260 residues of SEQ ID NO:2 and in the replacement at 279 and 285 residue places, for example, K258A, R259A, K260A, D279G and N285D change.Factor D has activated fB protease to the cutting of wild type fB.Thereby the fB3 that changes with its 5 amino acid combine with C3b but proteinase activity with minimum effectively.

FB1, fB2 and fB3 are the examples that can be used for the fB analog of practice of the present invention, but the invention is not restricted to these particular analog.Some embodiment of the present invention comprises any fB analog that suppresses complement pathway.In some embodiments, the fB analog is not fB1.In some embodiments, the fB analog is not fB2.In some embodiments, the fB analog is not fB3.In some embodiments, the fB analog comprises the one or more following amino acid whose one or more amino acid mutations corresponding to SEQ ID NO:2: 258,259,260,279,285,739,740,741,742,743,744,745 and 746 amino acids.Described one or more sudden change can be described amino acid whose replacement or disappearance or be close to it or add at least one amino acid within 1,2,3,4,5,6,7,8,9 or 10 amino acid.In some embodiments, listed amino acid whose function is upset, changes, strengthens or has suppressed in described interpolation, for example, (for example upset its (i) effect in another albumen of cutting, 740), (ii) conduct is by the effect (for example, 258,259 and/or 260) in the site of another albumen cutting, or (ii) its effect (for example, 279 or 285) in conjunction with another albumen.

Some embodiment of the present invention comprises with the aminoacid replacement that is selected from the group of being made up of alanine, glycine, valine, leucine and isoleucine one or more corresponding in 258,259 and/or 260 s' the amino acid.Some embodiment of the present invention comprises corresponding to one, two or three following amino acid whose disappearances: 258,259 and/or 260.Some embodiment of the present invention is included in next-door neighbour's amino acid whose at least one interpolation more than 1,2,3,4,5,6,7,8,9 or 10 corresponding to the amino place of 258,259 and/or 260 amino acids.

Some embodiment of the present invention comprises with 739 the amino acid of alanine replacement corresponding to SEQ ID NO:2.Some embodiment of the present invention comprises with being selected from 739 the amino acid of the aminoacid replacement of the group of being made up of alanine, glycine, valine, leucine and isoleucine corresponding to SEQ ID NO:2.Some embodiment of the present invention comprises the amino acid deletions corresponding to described 739 amino acids.

Some embodiment of the present invention comprises with being selected from 740 the amino acid of the aminoacid replacement of the group of being made up of glutamic acid, asparagine, alanine, serine, glycine and tyrosine corresponding to SEQ ID NO:2.Some embodiment of the present invention comprises with being selected from the aminoacid replacement of the group of being made up of valine, leucine, isoleucine, threonine, cysteine, methionine, aspartic acid, glutamine, phenyl alanine, tyrosine, tryptophan, glutamic acid, asparagine, alanine, serine, glycine and tyrosine corresponding to described 740 amino acid.Some embodiment of the present invention comprises the amino acid deletions corresponding to described 740 amino acids.

Some embodiment of the present invention comprises with being selected from 741 the amino acid of the aminoacid replacement of the group of being made up of tryptophan and alanine corresponding to SEQ ID NO:2.Some embodiment of the present invention comprises with described 741 amino acids of aminoacid replacement that are selected from the group of being made up of alanine, glycine, valine, leucine and isoleucine.Some embodiment of the present invention comprises with described 741 amino acids of aminoacid replacement that are selected from the group of being made up of tryptophan, tyrosine and phenyl alanine.Some embodiment of the present invention comprises the disappearance of described 741 amino acids.

Some embodiment of the present invention comprises with 742 the amino acid of glutamine replacement corresponding to SEQ ID NO:2.Some embodiment of the present invention comprises described 742 amino acids of aminoacid replacement with the group that is selected from glutamine, glutamic acid, aspartic acid and aspartic acid composition.Some embodiment of the present invention comprises the disappearance of described 742 amino acids.

Some embodiment of the present invention comprises with 743 and/or 745 the amino acid of phenyl alanine replacement corresponding to SEQ ID NO:2.Some embodiment of the present invention comprises aminoacid replacement described 743 and/or 745 amino acids with the group that is selected from phenyl alanine, tyrosine and tryptophan composition.Some embodiment of the present invention comprises one or more disappearance in described 743,744 and/or 745 amino acids.

Some embodiment of the present invention comprises with being selected from 746 the amino acid of the aminoacid replacement of the group of being made up of tryptophan and alanine corresponding to SEQ ID NO:2.Some embodiment of the present invention comprises with described 746 amino acids of aminoacid replacement that are selected from the group of being made up of alanine, glycine, valine, leucine and isoleucine.Some embodiment of the present invention comprises with described 746 amino acids of aminoacid replacement that are selected from the group of being made up of tryptophan, tyrosine and phenyl alanine.Some embodiment of the present invention comprises the disappearance of described 746 amino acids.

Some embodiment of the present invention is included in and is right after 739,740,741,742,743,744,745 and/or 746 amino acids places or inserts in these positions or replace amino acid more than 1,2,3,4,5,6,7,8,9 or 10.

Some embodiment of the present invention comprises with being selected from 279 the amino acid of the aminoacid replacement of the group of being made up of glycine, alanine and asparagine corresponding to SEQ ID NO:2.Some embodiment of the present invention comprises with described 279 amino acids of aminoacid replacement that are selected from the group of being made up of glycine, alanine, valine, leucine and isoleucine.Some embodiment of the present invention comprises with described 279 amino acids of aminoacid replacement that are selected from the group of being made up of aspartic acid, asparagine, glutamic acid and glutamine.Some embodiment of the present invention comprises the disappearance of described 279 amino acids.Some embodiment of the present invention comprises and is right after 279 amino acids or inserts in its position or replace amino acid more than 1,2,3,4,5,6,7,8,9 or 10.

Some embodiment of the present invention comprises with being selected from 285 the amino acid of the aminoacid replacement of the group of being made up of alanine and aspartic acid corresponding to SEQ ID NO:2.Some embodiment of the present invention comprises with described 285 amino acids of aminoacid replacement that are selected from the group of being made up of glycine, alanine, valine, leucine and isoleucine.Some embodiment of the present invention comprises with described 285 amino acids of aminoacid replacement that are selected from the group of being made up of aspartic acid, asparagine, glutamic acid and glutamine.Some embodiment of the present invention comprises the disappearance of described N285 amino acids.Some embodiment of the present invention comprises and is right after 285 amino acids or inserts in its position or replace amino acid more than 1,2,3,4,5,6,7,8,9 or 10.

Some embodiment of the present invention comprises 279,282,283,284 and 285 the one or more amino acid of replacement corresponding to SEQ ID NO:2.In some embodiments, substitute these amino acid with glycine, isoleucine, proline, histidine and aspartic acid respectively.

Some embodiment of the present invention comprises the sudden change to 258 as herein described, 259,260,279 and 285 amino acids.

In specific implementations, can use the factor B analog that comprises SEQ ID NO:4, one of 6 or 8 amino acid sequence (in case of necessity not with wherein contained burst) in the method for the invention.

Analog of the present invention comprises the factor B analog, and described factor B analog comprises the combination of replacement discussed in this article and kept fB1 discussed in this article, fB2 and/or the attribute of fB3 analog or any other fB analog.The present invention and then the fragment (the one or more territories that for example comprise the factor B with one or more amino acid changes as herein described) as these analogs is provided and has had the analog of the more than one attribute of analog as discussed above.In addition, described analog can comprise other aminoacid replacement, disappearance or insertion (for example, conserved amino acid replaces, the brachymemma of N end or C end etc.) thus the time described analog have at least 99.5%, 99%, 98%, 95%, 90%, 85%, 80%, 75% or 75% homogeneity.

In some embodiments, produce or carried dominant fB part with the level that approximates or surpassed the level of the natural fB that finds at local (for example, in retina) and C3b.In this, high level (being respectively 200 μ g/ml and 1,000 μ g/ml) that it should be noted that fB in the blood plasma and C3 may not can reflect the fB of (as retina) in other zone and the local horizontal of C3b.In some embodiments, produce or the level of the dominant fB part of carrying is in about 1%～about 100,000% of the level of the natural fB of local (for example, in the retina as animals such as the mankind) discovery and C3b; About 1%～about 10%; About 1%～about 20%; About 1%～about 30%; About 1%～about 40%; About 1%～about 50%; About 1%～about 60%; About 1%～about 70%; About 1%～about 80%; About 1%～about 90%; About 1%～about 100%; 90%～about 100%; 80%～about 100%; 70%～about 100%; 60%～about 100%; 50%～about 100%; 40%～about 100%; 30%～about 100%; 20%～about 100%; 10%～about 100%; 10%～about 20%; About 20%～about 30%; About 30%～about 40%; About 40%～about 50%; About 50%～about 60%; About 60%～about 70%; About 70%～about 80%; 80%～about 90%; About 100%～about 125%; About 100%～about 150%; About 100%～about 175%; About 100%～about 200%; About 100%～about 250%; About 100%～about 300%; About 100%～about 400%; About 100%～about 500%; About 100%～about 700%; About 100%～about 850%; About 100%～about 1000%; About 200%～about 300%; About 300%～about 400%; About 400%～about 500%; About 500%～about 600%; About 600%～about 700%; About 700%～about 800%; About 800%～about 900%; About 900%～about 1000%; About 250%～about 500%; About 500%～about 750%; About 750%～about 1000%; About 1000%～about 2000%; About 2000%～about 3000%; About 3000%～about 4000%; About 4000%～about 5000%; About 5000%～about 6000%; About 6000%～about 7000%; About 7000%～about 8000%; About 8000%～about 9000%; About 9000%～about 10,000%; About 10,000%～about 20,00%; About 10,000%～about 50,000%; Or about 50,000%～about 100,000%.

In order to carry out the rodent experiment, produce mfB1, mfB2 and mfB3 thereby in mouse fB, introduced with fB1, fB2 and the similar sudden change of fB3.At amino acid levels, people fB and mouse fB have 83% sequence homogeneity.Adorned amino acid is kept in mouse fB fully among the people fB.Although such sequence homogeneity is arranged, complement factor is brought into play function in the species specificity mode usually.All in vitro studies are undertaken by human specific test and the test of mouse specificity, and the rodent that at first choose fB mutant and mouse fB mutant have carried out in the body is studied.

Describe the exemplary process of 8 kinds of cDNA of generation (people's wild type fB and 3 dominant bodies and 4 similar muroid sequences) and the combination in carrier thereof hereinafter among theembodiment 8 in detail.

The inventor infers that the appropriate method of another reduction alternative route feedback loop is to disturb the function or the level of Complement Factor D (fD).Some embodiment of the present invention is with the dominant strategy fD function that is used to weaken.Below for example understand the factor D analog that is used to suppress to substitute the complement pathway activity.

Complement Factor D (fD) is cutting and the protease (Figure 12) that activates the fB in the C3bB compound.Factor D is present in the blood plasma with about 2 μ g/ml low-level usually and serves as catalyzer in the alternative route.In other words, so independent that the fD part combines, cuts fB with formation compound C3bBb, from this complex dissociation with C3bB, continue to repeat these steps then.According to expection, the fD that does not have proteolytic activity shows that the inactivation form can not suppress alternative route.In the presence of wild type fD, described dominant body makes the C3bB compound wait until wild type fD cutting simply in conjunction with also discharging.Yet the discovery of general introduction shows among the embodiment 21, and when not having the fB cutting, fD does not discharge or discharges from described compound with lower frequency/rate at least from described compound.Therefore, in some embodiments, dominant fD might serve as the inhibitor of alternative route actually.In some embodiments, dominant fD can be in conjunction with C3bB and is not dissociated or dissociate with the speed slower than wild type fD.Described compound will keep fB not cut and can not continue the process of complement activation by wild type fD.

Wild type people's factor D is provided among the SEQ ID NO:27.This is the preceding albumen of fD that has the signal peptide of crossing over 1～20 amino acids.

In some embodiments, the fD analog combines with the C3bB compound but does not cut fB or have the ability of the cutting fB of reduction.In some embodiments, compare fD analog of the present invention with wild type fD and comprise additional amino acid at the N end.For example, circulate in blood plasma with 1% the level that is lower than wild type fD with the fD variant of two additional amino acid Gly-Arg at the N end according to the show.This variant does not have proteinase activity and can only activate (Yamauchi etc., 1994 J Immunol.152 (7): 3645-53) by the trypsase of high concentration very.This dominant variant should become functional C3bBb (C3 convertase) and come the competition with wild type fD by combine and prevent it with C3bB.Therefore, the invention provides with wild type fD and compare the fD analog that comprises more than one additional amino acid at the N end, wherein said fD analog has ability or the speed of cutting fB reduction or that be eliminated.In some embodiments, this fD analog comprises and surpasses two extra N end amino acids.In some embodiments, this fD analog comprises 2,3,4,5,6,7,8,9 or 10 extra N end amino acids.In some embodiments, described extra N end amino acid comprises glycine and arginine.

In some embodiments, the factor D analog has the sudden change in the catalytic domain of wild type fD.The important component of the serine protease catalytic domain that three amino acid that it is believed that fD are fD.These three amino acid are corresponding to 66,114 and 208 amino acids of SEQ ID NO:27, also corresponding at Volanakis ﹠amp; 57,102 and 195 amino acids described in Narayana etc. " 1996 Protein Science 5:553-564 ".In some embodiments, can reduce or eliminate serine protease to one, two in these three amino acid or whole replacements but still can combine with C3B.In some embodiments, replaced amino acid with at least one neutral amino acid, amino acid that at least one is electronegative or at least one nonpolar amino acid corresponding to 66 amino acids of SEQ ID NO:27.In some embodiments, replaced amino acid with at least one charged amino acid or at least one nonpolar amino acid corresponding to 114 amino acids of SEQ ID NO:27.In some embodiments, replaced amino acid with at least one charged amino acid or at least one nonpolar amino acid corresponding to 208 amino acids.In some embodiments, with at least one aminoacid replacement that is selected from the group that alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenyl alanine, proline, serine, threonine, tryptophan, tyrosine and valine form corresponding to 66,114 or the amino acid of 208 amino acids of SEQ ID NO:27.

These fD analogs will be by combining and prevent with C3bB or slowing down its speed that becomes functional C3 convertase and come the competition with wild type fD.Therefore, some embodiment of the present invention comprises the fD analog that comprises corresponding to amino acid whose one or more sudden changes of amino acid His66, the Asp114 of SEQ ID NO:27 and Ser208.These three amino acid are also corresponding to adopting Volanakis﹠amp; His57, Asp102 and the Ser195 amino acid of used numbering in Narayana etc. " 1996 Protein Science 5:553-564 ".Described one or more sudden change can be aminoacid replacement or disappearance or the next-door neighbour or at 1,2,3,4,5,6,7,8,9 or 10 amino acid with at least one amino acid of interior interpolation.Some embodiment of the present invention comprises the fD analog of the sudden change that has the variation that causes fD serine protease catalytic domain, and wherein said fD analog still can combine with C3B, but described fD analog has the ability of the cutting fB of reduction.Analog of the present invention comprises the factor D analog that comprises as discussed above the combination that replaces and keep one or more attributes of fD analog as discussed above (as the ability of the cutting fB that reduces).The present invention and then analog as the fragment (the one or more territories that for example comprise the factor D albumen with one or more amino acid changes as herein described) of those analogs of the one or more attributes with analog that this paper discusses is provided.In addition, described analog can comprise extra aminoacid replacement, disappearance or insertion (for example conservative amino acid replaces, brachymemma of N end or C end or the like) thus described analog has at least 99.5%, 99%, 98%, 95%, 90%, 85%, 80%, 75% or 75% homogeneity.

The independent therapeutic agents that fD analog as herein described can activate as the blocking-up alternative route or be used in combination with other inhibitor of alternative complement pathway fD analog as described herein.Can as described, use fD analog as herein described, or substitute the application of fB as herein described with it to fB as herein described.

Factor H,factor H sample 1, MCP, DAF and CD59 work by suppressing complement activity usually.Some embodiment of the present invention comprises with factor H,factor H sample 1, CR1, MCP, DAF and/or CD59 (alone or in combination) regulates complement activity.Can use these albumen with its albumen form, or carry described albumen by nucleic acids encoding said proteins (for example, using carrier).In some embodiments, MCP, DAF or CD59 can regulate the soluble form of (for example, suppress) complement activity or solvable fragment (for example, wherein stride lose in the film district or replaced fragment).

To the relevant approach of complement or with the adjusting of various complement related conditions proteins associated

In some embodiments, the invention provides composition and the method that is used to regulate, adjust, suppress and/or strengthen complement approach relevant, that complement is relevant.The relevant approach of complement includes but not limited to CCP and agglutinin complement pathway and alternative complement pathway.In some cases, the relevant approach of complement may work in particular condition, one or more diseases.Therefore, some embodiment of the present invention provides adjusting, change, healing, inhibition, prevention, improve morbid state, slow down its progress and/or treat the method for described morbid state, and described morbid state is by the relevant approach mediation of more than one complement.These morbid states or the patient's condition include but not limited to that drusen forms, macular degeneration, AMD, xerophthalmia, ulcer of the cornea, atherosclerotic, BDR, vitreoretinopathy (Grisanti etc., Invest.Ophthalmol.Vis.Sci.32:2711-2717), the cornea inflammation, airway hyperreactivity, with Ia disease, the disease relevant with autoimmunity, lupus nephritis, systemic loupus erythematosus (SLE), arthritis (for example, rheumatic arthritis), rheumatism, antiphospholipid antibody syndrome, small intestine and kidney I/R damage, asthma, the atypia hemolytic uremic syndrome, II type membrano proliferative glomerulonephritis, the non-proliferative glomerulonephritis, pregnancy loss (for example, spontaneous pregnancy loss), glaucoma, uveitis, high intraocular pressure, brain damage (for example, traumatic brain injury), palsy (for example, see Arumugam etc., PNAS93 (12): 5872-6 (1996)), organ injury after the wound, organ injury (for example after the infarct, heart damage, nerve damage), vasculitis, ischemic damage and reperfusion damage, cerebrovas-cularaccident, Alzheimer's disease, graft rejection (for example, xenograft rejection or allosome suppress to repel), infect, septicemia, infectious shock;

Syndrome, myasthenia gravis, antibody-mediated skin disease, all antibody-mediated organ specificity diseases (comprise I type and type ii diabetes, thyroiditis, ITP and hemolytic anemia and DPN), multiple sclerosis, the cardiopulmonary bypass damage, polyarteritis nodosa, anaphylactoid purpura, serum sickness, Goodpasture, systemic necrotizing vasculitis, post-streptococcal glomerulonephritis, idiopathic pulmonary fibrosis (common interstitial pneumonia), membranous glomerulonephritis, myocarditis (for example, autoimmune myocarditis) (Kaya etc., Nat Immunol.2001; 2 (8): 739-45), myocardial infarction, muscular dystrophy (for example, lacking relevant muscular dystrophy), acute shock lung syndrome, adult Respiratory Distress Syndrome(RDS) with dystrophin, pour into again, the disease of repulsion and/or complement-mediated.

The formation of drusen may be with relevant as various diseases such as macular degenerations in the eye.In some cases, there is prompting to show that drusen forms and/or its correlation with disease relevant with complement activity (for example, seeing Fig. 1).Some embodiment of the present invention provides composition and the method that is used for regulating, adjust, suppress, reduce, hinder and/or reverse as the formation or the growth of animal drusens such as the mankind.For example, composition of the present invention or molecule can be transported to (for example, by being injected directly into (injection in the drusen) or intravitreal injection in the drusen) in the drusen.Thereby some embodiment of the present invention can be used for for example forming the progress of slowing down macular degeneration by suppressing drusen.

Atherosclerotic is usually directed to the relevant approach of complement according to the show, for example, see Niculescu etc., Immunologic Research, 30 (1): 73-80 (8) (2004) and Niculescu and Horea, Immunologic Research 30 (1): 73-80 (2004).Complement activation and C5b-9 deposition all can take place in the atherosis and experimental atherosclerosis the human artery usually.C5b-9 may cause lysis, and the inferior dissolubility assembly of C5b-9 is induced the activation and the propagation of smooth muscle cell (SMC) and endothelial cell (EC).The shortage of complement C6 has the protectiveness effect to the atherosclerotic of diet induced; this shows that the C5b-9 assembly is necessary or play an important role at least therein in the development of atherosclerotic lesion; for example; see Niculescu and Horea, ImmunologicResearch 30 (1): 73-80 (2004).Some embodiment of the present invention can be used for suppressing the formation of C5b-9 and/or suppress atherosclerotic.Thereby this formation and/or the activation that can suppress C5b-9 by formation or the step in the inhibition approach of direct inhibition C5b-9 realize.In some embodiments, factor B variant as herein described is administered to atherosclerotic site or potential site.This factor B variant has suppressed approach (for example, CCP and/or alternative complement pathway), and this formation or activation that has then suppressed C5b-9 or participated in atherosclerotic another complement pathway allied compound.The relevant albumen of complement that also has other participates in atherosclerotic, and the formation of described albumen and/or activation are suppressed in a similar manner or block.

Airway hyperreactivity (AHR) is the feature that includes but not limited to the various diseases of asthma (for example, allergic asthma).AHR is usually directed to the relevant approach of complement according to the show, for example, sees Taube etc., and 2006PNAS 103 (21): 8084-8089; Park etc., American Journal of Respiratoryand Critical Care Medicine 169:726-732, (2004); Thurman and Holers, No. the 20050260198th, JImmunology 176:1305-1310 (2006) and U.S. Patent bulletin.Park etc. have shown that the Crry-Ig that uses by intraperitoneal injection is influential to AHR.Some embodiment of the present invention provides and has been used for regulating, adjust, suppress, reduce, activate and/or increases composition and method as the AHR of animals such as the mankind.Can treat, alleviate, the concrete AHR diseases related that suppresses and/or improve includes but not limited to asthma, COPD (COPD), allergic bronchopulmonary aspergillosis, the hypersensitivity pneumonia, EC's property pneumonia, pulmonary emphysema, bronchitis, allergic bronchitis, bronchiectasis, cystic fibrosis, tuberculosis, the hypersensitivity pneumonitis, occupational asthma, sarcoid, RADS, interstitial lung disease, the HES, rhinitis, nasosinusitis, exercise-induced asthma, asthma is brought out in pollution, cough variant asthma, parasitic disease of lung, Respiratory Syncytial Virus(RSV) (RSV) infects, parainfluenza virus (PIV) infects, rhinovirus (RV) infects, Hantaan virus (Hantaan virus, for example, four jiaos of bacterial strains) infection and adenovirus infection.

Be usually directed to the relevant approach of complement as autoimmunity relevant disease, HLA-B27 related inflammatory disease, lupus nephritis and systemic loupus erythematosus immune correlated diseases such as (SLE) according to the show, for example, see Thurman and Holers, J Immunology 176:1305-1310 (2006).Lupus nephritis is a kind of complication of SLE.It is relevant with the self-immunprocess of lupus, the immune system antibody (antinuclear antibodies etc.) of body composition that creates antagonism in this self-immunprocess.The accumulation and cause inflammatory reaction in kidney usually of the compound of these antibody and complement.Some embodiment of the present invention provides and has been used for regulating, changes, cures, suppresses, prevention, improves and/or the method and composition of treatment immune correlated disease (for example, relate to as SLE etc. or about the immune correlated disease of complement pathway).

Arthritis is usually directed to the relevant approach of complement according to the show, for example, sees Thurman and Holers, J Immunology 176:1305-1310 (2006) and Banda etc., J Immunol.177 (3): 1904-12 (2006).Alternative complement pathway plays an important role in inducing arthritis even may be essential.Some embodiment of the present invention provide be used to regulate, change, healing, inhibition, prevention, improvement and/or the treatment of arthritis method and composition of rheumatic arthritis or inflammatory arthritis for example.

Glaucoma relates to the one group optic nerve disease of retinal ganglial cells with the characteristic pattern forfeiture of optic neuropathy.About 25% the glaucoma patient that has the retinal ganglial cells forfeiture has normal intraocular tension.High intraocular pressure (OHT) is the glaucomatous material risk factor of development, and reduces the main method that high intraocular pressure is present glaucoma treatment by medicine or operation.High according to the show intraocular pressure is usually directed to the relevant approach of complement with glaucoma, for example, sees Khalyfa etc., Molecular Vision, 13:293-308 (2007); Stasi etc., IOVS 47 (3): 1024-1029 (2007); With Kuehn etc., Experimental Eye Research 83:620-628 (2006).C1q and C3 more highland expression and/or existence in suffering the retina of OHT according to the show.Some embodiment of the present invention provides and has been used for regulating, change, cure, suppress, prevent, improve and/or treating glaucomatous method and composition.

Uveitis is relevant with complement pathway usually according to the show, for example, sees Mondino and Rao, Investigative Ophthalmology ﹠amp; Visual Science 24:380-384 (1983) and Jha etc., Molecular Immunology 44:3901-3908 (2007).Mondino and Rao find, the tested complement component of all in the aqueous body fluid increases in the patient that the operated eye history is arranged before this mean value of determination of serum and suffering among the anterior uveitic patient the highest.Some embodiment of the present invention provides and has been used for regulating, change, cure, suppress, prevent, improve and/or treating uveitic method and composition.

BDR is one of main reason of visual loss in the middle aged individuality.The activation that it is believed that complement system in the pathogenesis of BDR, play an important role (for example, seeing Jha etc., Molecular Immunology 44:3901-3908 (2007)).Some embodiment of the present invention provides the method and composition that is used to regulate, change, cure, suppress, prevent, improve and/or treat BDR.

Proliferative vitreoretinopathy (PV) is one of most common complication of retinal detachment.PV is relevant with complement activity, for example, sees Grisante etc., Invest Ophthalmol Vis Sci.1991; 32 (10): 2711-7 and Grisante etc., Ophthalmologe.1992; 89 (1): 50-4.Some embodiment of the present invention provides the method and composition that is used to regulate, change, cure, suppress, prevent, improve and/or treat PV.

Antiphospholipid antibody syndrome, small intestine and renal ischaemia (for example pour into I/R damage, atypia hemolytic uremic syndrome, II type membrano proliferative glomerulonephritis and pregnancy loss more according to the show, spontaneous pregnancy loss) is usually directed to the relevant approach of complement, for example, see Thurman and Holers, JImmunology 176:1305-1310 (2006).

Brain damage (for example, traumatic brain injury) is usually directed to the relevant approach of complement according to the show, for example, sees Leinhase etc., J Neuroinflammation 4:13 (2007) and BMC Neurosci.7:55 (2006).Leinhase time dependent systemic complement activation occurred behind the experimental traumatic brain injury that was presented at wild type (fB+ /+) mouse in 2006.By contrast, the degree of systemic complement activation fB-/-mouse in significantly the reduction.Some embodiment of the present invention provides and has been used for regulating, changes, cures, suppresses, prevents, improves and/or treatment nerve cell death, traumatic neurotrosis (for example, brain damage), the neural inflammation of complement-mediated and/or the method and composition of neuropathology.

Ischemic damage and reperfusion damage can make by the generation of the various compounds that may be harmful to of cell and tissue generation or oxidation increase, and this may cause the oxidative damage of pair cell and tissue or cause its death.For example, the renal ischaemia reperfusion injury can cause the histology infringement to kidney, comprises the feature of tubular injury and change acute tubular necrosis.The renal dysfunction that is caused makes usually and can be gathered by the nitrogenouz wastes of renal excretion such as serum urea nitrogen (SUN) etc.Ischemic damage and reperfusion also may cause the damage such as distant place organs such as lungs.Some embodiment of the present invention for example to suffer from ischemic damage and reperfusion or be in stand or develop adopted when animal in the danger of ischemic damage and reperfusion is used complement pathway as instrumentalities such as inhibitor (for example, the inhibitor of factor B activity).In some embodiments, these instrumentalities can prevent, weaken or suppress because at least one symptom of the damage that ischemic damage and reperfusion causes.Can include but not limited to the ischemic damage and reperfusion damage of heart ischemia reperfusion injury (as myocardium infarct or coronary artery bypass surgery etc.), central nervous system ischemic damage and reperfusion damage, four limbs or finger/toe with the ischemic damage and reperfusion damage of method and composition of the present invention prevention or other type that weakens, as the ischemic damage and reperfusion damage of internal organs such as lung, liver or small intestine or the ischemic damage and reperfusion damage of any transplanted organ or tissue.For example see that the PCT that myocardial infarction and complement pathway are discussed discloses WO03/061765 number.

Inflammation is main teiology decisive factor (Ridker, 2007 Nutr.Rev.65 (12 Pt 2): S253-9) of myocardial infarction.Caused improving (Wollert, 2008, the Curr.Opin.Pharmacol.1 month 31 [Epub]) of contractile function according to another the conveying that is presented at marrow (doing) cell behind the acute myocardial infarction AMI (for example, carrying in the coronary artery).In addition, stem cell can make the cardiac muscle regeneration (Orlic etc., 2003 Pediatr.Translpant.7 Suppl 3:86-88) of infarct.Mescenchymal stem cell provides Cardioprotective effect (Guo etc., 2007 Inflammation30 (3-4): 97-104) in ischemic heart disease according to the show.In the present invention, can be by carrying stem cell as any ways such as (for example, being expelled in ill cardiac muscle and/or the healthy cardiac muscle (for example adjacent healthy cardiac muscle)) of directly injection in intracoronary injection, the cardiac muscle with affected area.In some embodiments, thus handling mammal with cell factor makes described stem cell can run to the cardiac muscle of infarct its stem cell is mobilized in the circulation.

Various cells are used for various application in vivo.The problem that stem cell is used in vivo is that for example the survival in interesting areas and/or inoculation are lower than expection to described stem cell.The inventor believes an inflammation that remarkable reason is described site of low inoculation and the low survival of stem cell.Therefore, the invention provides methods of treatment and/or improve stem cell survival and/or the method for inoculation, described method be included in use or mobilize before the stem cell, during and/or use composition of the present invention afterwards.In some embodiments, complement inhibitor of the present invention serves as antiinflammatory, and described antiinflammatory will create favorable environment so that stem cell guiding and survive in (for example, impaired heart) and therefore reparation or alternative damaged tissues in required the inoculation zone in.Stem cell can be used in the solution that also comprises as molecules of the present invention such as fB analog or fD analogs.Stem cell can be but be not limited to candidate stem cell, embryonic stem cell, mescenchymal stem cell, neural stem cell, mammary gland stem cell, sense of smell stem cell, pancreatic stem cell, myeloid-lymphoid stem cell, multipotential stem cell or pluripotent stem cell.Described stem cell can be autologous stem cells, allogeneic stem cell or homogenic stem cell.

According to showing that complement activity and muscular dystrophy (for example, lacking relevant muscular dystrophy with dystrophin) are relevant.For example, see that PCT discloses WO2007130031 number, Spuler ﹠amp; 2001 Neurology 56:1472-1481 such as Engel 1998Neurology 50:41-46 and Selcen.Therefore, some embodiment of the present invention provides the method and composition that is used to regulate, change, cure, suppress, prevent, improve and/or treat muscular dystrophy.

Complement activity can promote the cornea inflammation.Therefore, some embodiment of the present invention provides the method and composition that is used for (for example, after operation) adjusting, change, healing, inhibition, prevention, improvement and/or treats the cornea inflammation.In some embodiments, molecule of the present invention is used by eye drops or alternate manner as herein described.

Some embodiment of the present invention provides the method for the effect that is used to strengthen coronary artery or Peripheral arteries Coronary Artery Bypass or postangioplasty.In some embodiments, with coding albumen of the present invention (for example, fB1 and/or fB3) the carrier of the present invention vascular cell (for example, endothelial cell) of transduceing.In some embodiments, vascular cell was transduceed before being transplanted to animal.In some embodiments, vascular cell is positioned at body.

Alleviation to pain, suffering and the inflammation of postoperative patient is the field of special concern in the clinical medicine, particularly along with the increase of the quantity of the outpatient operation that carries out every year.Composition of the present invention can be used for for example coming inflammation-inhibiting by suppressing complement activity.Therefore, composition of the present invention can be used to alleviate for example inflammation of postoperative patient.In some embodiments, with composition of the present invention is local carry (for example, peri-operation period is carried) to the operation site so that inflammation-inhibiting, this described in some cases composition will ease the pain and suffering.In some embodiments, composition of the present invention (for example, in physiological electrolyte carrier fluid) in solution is used.In some embodiments, described composition is delivered directly to surgical site with the irrigation solution that contains described composition by the peri-operation period conveying.In some embodiments, because local peri-operation period carrying method of the present invention, can adopt as the required result of treatment of medicament acquisition of intravenous, intramuscular, the necessary dosage of other carrying method such as subcutaneous and oral with being lower than.In some embodiments, when peri-operation period uses, described solution will cause significantly reducing clinically the pain and/or the inflammation of surgical site, thereby make the patient reduce and allow in due course the activity in corrective surgery site more early to the demand of postoperative antalgesic (for example, opiate).In some embodiments, for surgeon and operating room personnel's role, the use of comparing solution of the present invention with conventional irrigating solution does not need to expend the extra time.In some embodiments, composition of the present invention (for example, in irrigating solution) is used for arthroscope, cardiovascular and common vascular treatment program and diagnostic program, urological department program, general surgery's surface of a wound and the general surface of a wound.Can by but be not limited to injection (for example, through syringe), apply conveying composition of the present invention in agent (as solution, emulsifiable paste or gel etc.) via irrigating solution, as the part of the bandage on the surface of a wound or in the part.

μ g in another embodiment can be with composition of the present invention or molecule and LUCENTIS

Perhaps use in conjunction with the molecular combinations of VEGF or inhibition angiogenesis.LUCENTIS

Be used for the treatment of moist AMD.Some embodiment of the present invention also can be used for the treatment of moist AMD.Therefore, the invention provides the method and composition that is used for the treatment of moist AMD, described method comprises uses LUCENTIS

With composition of the present invention, wherein they can be used separately or together.In addition, intraocular inflammation is to use LUCENTIS

Back institute report one of modal bad reaction, for example, see LUCENTIS

" fully prescription information ".Therefore, the invention provides and be used for suppressing or reduce inflammation of eye section (for example, by using LUCENTIS

The inflammation of eye section that causes) method, described method are included in uses LUCENTIS

Simultaneously and/or use molecule of the present invention or composition afterwards before.

Can use as the whole bag of tricks of a part of the present invention and/or composition to come to promoting and/or causing that the complement pathway of disease regulates, adjusts, suppresses and/or activate.Some embodiment of the present invention adopts albumen to come the adjusting approach.

To using of animal and/or cell

This paper provides and has regulated relevant composition and the method for the relevant approach of complement.This can or finish in the body of external, earlier external back in vivo.Can finish using by any way for example described herein or known in the art to animal.In some embodiments, by the nucleic acid of using encoding proteins described albumen " indirectly " is used.In some embodiments, albumen itself is used animal.Those skilled in the art understand and the carrying method that delivery of composition of the present invention is matched to the expectation site in the animal.

In some embodiments, can be with composition (composition that for example, comprises analog) local application or systemic application.Useful route of administration is as described herein and be known in the art.Introducing method or application process include but not limited to intracutaneous, intramuscular, intraperitoneal, intravenous, subcutaneous, in the nose, in the tracheae, local (topical), suck, transdermal, rectum, the outer approach of stomach and intestine, epidural, encephalic, in the brain, in the ventricles of the brain (intraventricular), under the dura mater, in the joint, in the sheath, intracardiac, in the coronary artery, in the vitreum, under the retina, in the camera oculi anterior, especially (particular), local on the cornea, under the conjunctiva, capsula bulbi is injection down, by applying eye drops, oral route, through balloon catheter, through support or its any combination.In some embodiments, composition of the present invention or molecule are for example used drusen by being injected directly into to come in the drusen.Systemic application can be to be not limited to by injection or by saturating mucous membrane and/or transdermal delivery.In some embodiments, composition of the present invention at first can be led other site except that disease site for example.For example for for the AHR that takes place in the animal lung, the intraperitoneal injection of albumen of the present invention or nucleic acid may cause the variation of AHR in the lung, for example, sees Park etc., American Journal of Respiratory andCritical Care Medicine 169:726-732, (2004).In some embodiments, the dosage level of composition and/or mode of administration may depend on the character of the character of said composition, the patient's condition to be treated and/or the situation of individual patients.In some embodiments, used the cell of expressing albumen of the present invention.These cells can be cell-line, heterogenous cell, homogeneous variant cell or autogenous cell.

(for example comprise embodiment) in some embodiments to ocular administration, with molecule of the present invention or carrier approximately weekly, every month, per February, per March, per June, per September, every year, per 18 months, per 2 years, per 30 months, per 3 years, per 5 years, per 10 years uses once or as required and uses. (for example comprise the embodiment to ocular administration) in some embodiments, with molecule of the present invention or per approximately 1 week～4 week of carrier, per approximately 4 week～8 weeks, per approximately 1 week～April, per approximately March～June, per approximately April～August, per approximately June～December, per approximately September～15 months, per approximately December～18 months, per approximately 15 months～21 months, per approximately 18 months～24 months, per approximately 1 year～2 years, per approximately 1.5 years～3 years, per approximately 2 years～4 years, per approximately 3 years～5 years, per approximately 5 years～7 years, used in per approximately 7 years～10 years or per approximately 10 years～20 years.

(for example comprise embodiment) in some embodiments, molecule of the present invention or carrier are used more than 1,2,3,4,5,6,7,8,9 or 10 time at it throughout one's life to the patient ocular administration.(for example comprise embodiment) in some embodiments, slow virus carrier of the present invention is used more than 1,2,3,4,5,6,7,8,9 or 10 time at it throughout one's life to the patient ocular administration.

In some embodiments, can be with antiinflammatory agent and molecule of the present invention or carrier (for example, fB3) combination transfer.Can be before using molecule of the present invention or carrier, simultaneously and/or carry antiinflammatory agent afterwards.In some embodiments, antiinflammatory agent is used in solution identical with molecule of the present invention or carrier place and/or syringe.In some embodiments, molecule of the present invention or carrier and antiinflammatory agent are administered to eye jointly, for example, as described herein.

Many antiinflammatory agents are known in the art and include but not limited to sodium sulfo benzoate, dexamethasone sodium phosphate, fluorometholone, bromfenac, pranoprofen, RESTASIS between dexamethasone, dexamethasone^TM, cyclosporin ophthalmic emulsion, naproxen, glucocorticoid, ketorolac, brufen, Tolmetin, NSAID (non-steroidal anti-inflammatory drug), steroidal anti-inflammatory medicine, Diclofenac, Flurbiprofen, Indomethacin and suprofen.

Some embodiment of the present invention comprises the albumen that suppresses complement activity and/or the albumen that suppresses complement activity is carried out encoded carrier.Some embodiment of the present invention comprises using the carrier of albumen and this albumen of coding or the another kind of albumen of the present invention of encoding.Can be before using carrier of the present invention, simultaneously and/or carry albumen of the present invention afterwards.In some embodiments, albumen of the present invention is used in solution identical with carrier of the present invention place and/or syringe.In some embodiments, albumen of the present invention and carrier of the present invention are administered to eye jointly, for example, as described herein.

Nucleic acid

In order to ensure the part and the long-term expression of interested nucleic acid, some embodiment of the present invention considers to use nucleic acid or carrier to come transformant.Should not think that the present invention is subject to any specific nucleic acid carrying method, and in can be in practice of the present invention with body or external nucleic acid conveyance strategy use any available nucleic acid to carry to carry a device, or use controlled cell (as the technology of Neurotech, Lincoln, RI, for example, see United States Patent (USP) the 6th, 231, No. 879, the 6th, 262, No. 034, the 6th, 264, No. 941, the 6th, 303, No. 136, the 6th, 322, No. 804, the 6th, 436, No. 427, the 6th, 878, No. 544) and use coding to treat the nucleic acid (for example, " naked DNA ") of albumen itself.The present invention can use as various conveyings such as carriers and carry device.For example, slow virus, adenovirus vector (are for example seen United States Patent (USP) the 7th, 045, No. 344), the AAV carrier (for example sees United States Patent (USP) the 7th, 105, No. 345), plasmid (for example sees United States Patent (USP) the 6th, 936, No. 465), other viral vectors (for example, herpes virus, United States Patent (USP) the 5th, 830, No. 727 and the 6th, 040, No. 172; The fourth hepatovirus, United States Patent (USP) the 5th, 225, No. 347; With EBV virus, United States Patent (USP) the 6th, 521, No. 449), amphitrophic lipid, as cationic polymers such as polymine (PEI) and polylysines, penetrate shape (combburst) molecule and star as comb and penetrate other nucleic acid mode of dendrimer, nonionic lipid, anion lipid, vesica, liposome and gene transfer such as shape (starburst) molecule and (for example see, United States Patent (USP) the 6th, 958, No. 325 and the 7th, 098, No. 030; And Langer, Science 249:1527-1533 (1990); Treat etc. are in " Liposomes " in " The Therapy of Infectious Disease and Cancer "; With Lopez-Berestein ﹠amp; " Liss, New York, 317 pages-327 pages and 353 pages-365 pages (1989) " that Fidler writes); " naked " nucleic acid etc. can be used for practice of the present invention.Only for example purposes, some discussion of this paper will concentrate on some specific support type, comprise that BIV is the slow virus far away with the HIV relation as the slow virus carriers such as slow virus carrier from BIV (BIV) exploitation and acquisition.

Carrier is the mode that is used for introducing at interested target cell interested nucleic acid (for example, treatment nucleic acid for example, can be encoded and treat the nucleic acid of albumen).Usually if can carry alien gene or transgenosis and can enter the target cell and the initiation materials such as nucleic acid of expressing therein make up or obtain carrier from all.The suitable initiation material that can be used to obtain carrier comprises transposons known in the art, plasmid, virus, PCR product, cDNA and mRNA or the like.The method that is used to obtain or make up interested carrier includes but not limited to standard gene operating technology known in the art, sequencing reaction, restriction enzyme, polymerase, PCR, PCR soeing (PCR Splicing by OverlappingExtension), connection, recombinase reaction (for example, the GATEWAY of Invitrogen

Technology), on nucleic acid activated other enzyme, bacterium and virus multiplication material and method, chemical substance and reagent, rite-directed mutagenesis scheme or the like, for example see the article of Maniatis etc. " Molecular Cloning ".

Except optional marker gene, carrier of the present invention can also carry various so-called genetically modified nucleotide sequence, and they can for example be used as selection approach or be used for strengthening expression.In some embodiments, thus the foreign nucleus nucleotide sequence should have enough sizes to make and to produce the virion of surviving.For example, some virion is only packed the nucleic acid of specific dimensions scope.These genetically modified incomplete tabulations for example comprise, coding is as the sequence of albumen such as single-chain antibody, antibody and various antigen combining forms thereof, ribozyme, as inhibitory RNA molecules such as siRNA, catalytic antibody and antisense sequences.

Albumen can be the albumen of treatment albumen or influence treatment albumen.In addition, albumen can be intact proteins or its functional activity fragment.Described albumen can be for example participate in or the albumen of regulating inflammation (for example, wherein said albumen is treatment albumen), thus maybe can be to regulate the albumen of inflammation by for example acting on complement factor, act on gene or its instrumentality of expressing complement factor or acting on another gene that can regulate the element that causes inflammation.

In some embodiments, the sequence of being expressed is connected with the promotor navigability.In some embodiments, the transgenosis insert is connected with the second promotor navigability.

For construct disclosed herein, the selection of promotor is fully in those skilled in the art's technology category, and the selection of promotor extends to any eukaryotic promoter, prokaryotic promoter or viral promotors that can guide the genetic transcription in target or the host cell according to the present invention, described target or host cell transform (for example has first promotor of use, functional promotor in producing cell) construct or use the construct of second promotor (for example, the functional promotor in final target cell).Promotor can be for example tissue-specific promoter, cell specificity promotor, inducible promoter, repressible promoter, synthetic promoter or hybrid promoters.Can in construct of the present invention, settle and surpass a promotor.The example that can be used on the promotor in the construct of the present invention includes but not limited to phage (PL) promotor; The SV40 early promoter; Herpes simplex virus (HSV) promotor; As people CMV is cytomegaloviruses such as early promoter (CMV) promotor; Be subjected to promotor (tet) system of the trans-activating response of tetracycline control; As long terminal repeat (LTR) promotors such as MoMLV LTR, BIV LTR or HIV LTR; The U3 district promotor of Moloney muroid sarcoma virus (Moloney murine sarcoma virus); Granzyme A (Granzyme A) promotor; The adjusting sequence of metallothionein gene; The CD34 promotor; The CD8 promotor; Thymidine kinase (TK) promotor; B19 parvovirus promotor; The PGK promotor; The glucocorticoid promotor; As heat shock protein (HSP) promotors such as HSP65 and HSP70 promotors; Immunoglobulin promoter; The MMTV promotor; Rous sarcoma virus (RSV) promotor; The lac promotor; The CaMV35S promotor; With the nopaline synthase promoter.Many promotors can from as Stratagene (La Jolla, Calif.) and Invitrogen (Carlsbad, CA) etc. commercial source obtains.

In some embodiments, promotor comprises the promoter region of LTR, as 5 ' LTR promotor of HIV or BIV etc.In some embodiments, promotor comprises CMV promotor and PGK promotor.In some embodiments, promotor is MND promotor (Robbins etc., 1997, J.Virol.71:9466-9474) or as the MNC promotor of MND promotor derivative, in the MNC promotor, LTR enhancer and minimum CMV promotor are merged (Haberman etc., J.Virol.74 (18): 8732-8739,2000).

The heterologous intron is known and the example includes but not limited to people's betaglobulin gene intron.In some embodiments, carrier of the present invention comprises intron, for example, and as the gene of encoding proteins or the intron of an interested genetically modified part.In some embodiments, used intron can obtain from SV40 virus or human insulin gene in some construct of the present invention.In some embodiments, in the retroviral construct body, intron will be positioned at the upstream of gag and/or pol code area.

Burst or targeting sequencing are known and can be used in the expression construct, for example are used for expressing as albumen of the present invention such as fB analog or fD analogs.Burst is translated in framework and the terminal peptide that links to each other of the aminoterminal of the polypeptide of choosing, thereby secretory signal sequence is by causing the secretion of described polypeptide with mechanism's interaction of host cell.As the part of described secretion process, this secretory signal sequence will be cut off.PLAP's secretory signal sequence is the example of burst.The present invention is not subjected to the restriction of specific secretory signal sequence and other burst well known by persons skilled in the art.Term " burst " also refer to the to encode nucleotide sequence of secretion peptide.If comprised burst, then this burst can be native sequences, homologous sequence or heterologous sequence.

Viral gene or genetically modified expression are usually directed to the suitable promotor that is connected with the nucleic acid sequence encoding navigability.Term " navigability connection " or " maneuverability connection " are interchangeable and refer to element arrangements in the construct that wherein composition is configured so that carry out its function common, expectation or regulation.Promotor or other control element needn't link to each other with coded sequence.For example, the residue of intervention can be arranged between promotor or control element and code area, as long as functional relationship is held.

As disclosed herein, can further comprise 3 ' the polyadenylation signal (polyA) that is positioned at coded sequence according to one or more constructs of the present invention.The polyA afterbody can be for being enough to promote for example virtually any size of the stability in cytoplasm.The polyA signal can be derived from as slow viruss such as BIV, HIV and SIV.Yet the polyA sequence also can be derived from other cell or virus, as is derived from SV40 etc.Known in this area, a lot of polyA sites are known and can be used as design alternative, perhaps can use synthetic polyA.

In some embodiments, therapeutic gene can be to express complement factor or its instrumentality or resist the generation of the element that promotes inflammation or the gene of function, such as ribozyme, catalytic antibody, inhibitory RNA, antisense molecule or the like.

Viral vectors

The invention is not restricted to the specific virus carrier.Viral vectors includes but not limited to that retroviral vector, slow virus carrier, adenovirus vector (for example see United States Patent (USP) the 7th, 045, No. 344), the AAV carrier (for example sees United States Patent (USP) the 7th, 105, No. 345), (for example, United States Patent (USP) the 5th, 830 for herpesvirus vector, No. 727 and the 6th, 040, No. 172), fourth hepatovirus carrier (for example, United States Patent (USP) the 5th, 225, No. 347), SV40 carrier and EBV carrier (for example, United States Patent (USP) the 6th, 521, No. 449).

Can with virion of the present invention (for example, based on BIV carrier) in vivo or external pair cell (for example, mammalian cell) use.Carrier (viral vectors and non-virus carrier) can be used for transduceing or transformant, described cell includes but not limited to neoblast, noble cells, somatic cell, initial cell and/or stem cell.In some embodiments, intention is used for stem cell the people is used and be not used in the women who is transplanted to suitable false pregnancy so that differentiation and growth are the baby.

In some embodiments, virion is encoded for allos (with respect to virus and/or target cell) code area or transgenosis.In some embodiments, described allos code area coding treatment product.Some virion that generate according to the present invention are but the particle that is not limited to recombinate, recombinant virus particle, reorganization BIV particle, recombinant vector particle or virion." reorganization particle or virion " is meant the virion that contains based on the vector nucleic acid of virus.In some embodiments, the vector construction body can be included in and (for example be derived from virus, the virus that is different from BIV) in the particle, for example, retrovirus or slow virus such as FIV, HIV, SIV, BIV and EIAV etc., the strain of described Virus Type can openly be used to serve as the initiation material that is used to obtain interested carrier.

Usually, can by at first through centrifugal (for example, the centrifugal medium that contains virus) thus collect virion and remove the concentration that supernatant increases some virion of the present invention then.In some embodiments, remove major part or all substantially supernatant and virion is suspended in more in the small size.Thereby some embodiment comprises being suspended in the little 10 times liquid of the initial volume of volume ratio before centrifugal by the virus that will collect virus is concentrated 10 times.In some cases, this can cause transduction efficiency to increase by 3 times～10 times.One skilled in the art will readily appreciate that and to cause the higher increase of transduction efficiency virus is further concentrated.Can use separately or include but not limited to tangentially flow purifying, diafiltration, ion-exchange chromatography, affinity chromatography, size exclusion chromatography, immune affinity chromatographic, reverse-phase chromatography, heparin sepharose affinity chromatography and other separation known and conc forms with other conc forms that other form as herein described is used in combination.In some embodiments, virion is concentrated about 1.5 times～about 1000 times, about 2 times～about 90 times, about 2 times～about 80 times, about 2 times～about 70 times, about 2 times～about 60 times, about 2 times～about 50 times, about 2 times～about 40 times, about 2 times～about 30 times, about 2 times～about 20 times, about 2 times～about 15 times, about 2 times～about 10 times, about 2 times～about 7 times, about 2 times～about 5 times, about 5 times～about 500 times, about 10 times～about 500 times, about 50 times～about 500 times, about 100 times～about 500 times, about 200 times～about 500 times, about 300 times～about 500 times, about 400 times～about 500 times, about 450 times～about 500 times, about 500 times～about 1000 times, about 600 times～about 1000 times, about 700 times～about 1000 times, about 800 times～about 1000 times, about 900 times～about 1000 times, about 10 times～about 200 times, about 50 times～about 300 times, about 50 times～about 150 times, about 50 times～about 100 times, about 100 times～about 200 times, about 100 times～about 300 times, or about 300 times～about 500 times.

Biv vector is herein as the example of exemplary carrier type and nucleic acid carrying method/composition and the example appearance that can be used for retrovirus of the present invention or slow virus carrier.Yet, the invention is not restricted to biv vector, even be not limited to slow virus carrier or retroviral vector.Biv vector only is some examples of carrier that can be used according to the invention with the system that produces it.Other carrier also can be used for the present invention.For the characteristic that this paper describes biv vector, can be with corresponding characteristics design in other carrier (as other retrovirus under other slow virus and HIV virus and some situation).Therefore, BIV is provided as illustrative embodiments.

Still do not know BIV and can cause human diseases.Yet the biv vector multiple human cell that can transduce comprises as eye cells such as RPE cells.In some embodiments of the present invention, carrier only comprises the required minimum BIV element (Molina etc., 2004) of transfer therapeutic gene.Nearly all viral gene in these carriers can be removed, described viral gene accounts for that BIV is genomic to surpass 90%.The characteristic of some biv vector system includes but not limited to: high titre, for example, at least 106 and up to 3 * 10⁹Above transduced unit/ml; Effective outer-gene in the wide spectrum human cell shifts; Effective gene in retina cell's the body is shifted; Equal or exceed the extensive security feature of other slow virus carrier system in some embodiments; And the technology that is used for expansion scale and manufacturing.In for example Matukonis etc., 2002; Molina etc., 2002; 2004; United States Patent (USP) the 6th, 864 No. 085, the 7th, 125, No. 712 and the 7th, 153, has been described the example of BIV system in No. 512.

Can use a plurality of different combination of DNA construct obtain to hold interested therapeutic gene, carry genomic BIV particle based on virus.In an individual system, 4 kinds of DNA compositions are used for biv vector production.These DNA compositions comprise the expression construct of coding biv vector sequence; The expression construct of coding BIV rev sequence; The expression construct of coding BIV gag/pol sequence; Expression construct with the coding coating.In some embodiments, the coating code area is not to be derived from BIV.The expression construct of code carrier sequence may produce the required genetically modified RNA that carries that is packaged in carrier/virion.The expression construct of gag/pol and coating has produced the BIV capsid protein that forms described carrier granular.The expression construct of rev has produced assists vector rna is transported out nuclear albumen in addition.Rev can be placed on gag/pol construct and/or the env construct.In some embodiments, rev is not the part of gag expression construct, pol expression construct or env expression construct.In some embodiments, can use to have carried to be incorporated into the cell-line that host cell is the more than one BIV gene in the genome, so that for example when introducing biv vector, make the number minimum of transformation event.In some embodiments, can eliminate needs when preparing carrier with having merged the cell-line that produces the necessary all the components of described carrier to transforming.Some method that is used to produce carrier granular of the present invention relates to described expression construct cotransformation in the cell of tissue culture.As package carrier RNA in cytoplasm and after assembling described particle, thus the carrier granular permeate through cell membranes sprout and obtained the lipid bilayer shell, and in described tissue culture medium (TCM), gather, can be where necessary with them from described tissue culture medium (TCM) purifying and/or concentrate.At for example Rigg etc., Virology218:290-295 is described in (1996) by the method for the virion that is generated by transformant in collection.

The embodiment of slow virus carrier or retroviral vector (for example about) in some embodiments can incorporate part gag gene from virus genomic dna fragmentation into.For example, BIV gag coded sequence is about 1431 nucleotide usually.In some embodiments, a part that is used in the gag code area in the interested carrier will comprise about 102 nucleotide of being no more than of described gag code area.In some embodiments, a part that is used in the gag code area in the interested carrier will comprise about 76～about 500, about 76～about 200, about 76～about 102, about 76～about 100, about 76～about 95, about 76～about 90, about 76～about 85, about 76～about 80, about 80～about 90 or about 90～about 100 nucleotide of described gag code area.In some embodiments, this RNA sequence can be promoted vector rna is packaged in the carrier granular.In some embodiments, dna fragmentation can comprise be selected from by vif, vpw, vpy, tat, vpu, vpr, nef, tmx or rev (as from BIV, HIV or other slow virus etc.) thus the gene or the code area of the albumen of the group of forming are used to promote gene transfer.

The biv vector construct can and then comprise more than one regulating element, as the rna transport element (for example, rev response element (RRE)), composing type transhipment element such as Mason-Pfizer monkey disease poison CTE or avian leukosis virus CTE etc., strengthen the sequence of translation, burst, the copy number control element, integrate the compatibility sequence, terminator sequence, the mRNA targeting sequencing, scaffold attached region (SAR) (for example, being derived from human SAR), usually at the poly-purine passage of 3 ' LTR upstream, posttranscriptional regulatory element is (as the posttranscriptional regulatory element (WPRE of WCHV; Zufferey etc., Virol.73 (4): 2886-92 (1999)) etc.), internal ribosome entry site (IRES), ribosome bind site (RBS), enhancer and known in the art other are regulated sequence.In some embodiments, transgenosis (for example, the transgenosis that is connected with the second promotor navigability) is positioned at the BIV RRE downstream of supposition.RRE can be from other slow virus except that BIV.For the most of genetic elements that comprise in the interested construct, described element is configured in the mode that produces the navigability expression product and is connected, that is, described element is connected by navigability ground.Can be from other nucleic acid or, purchase synthetic and/or the described element of subclone from natural origin etc.

In some embodiments, retroviral vector construct body of the present invention or slow virus carrier construct are from the inactivation carrier.For example, when having LTR, the part in the U3 district of 3 ' LTR of biv vector construct may lack or be substituted by heterologous sequence.In the case, transgenosis can be connected with the internal promoter navigability.In some embodiments, the U3 element can and then contain the sequence of promoting polyadenylation.For example, can substitute the part (for example, seeing Sehet etc., Mol.Cell Biol., 12:5386-5393 (1992)) in the U3 district of 3 ' LTR with SV40 polyadenylation signal in late period enhancer element.

" packing construct ", sometimes be called the assisting building body, be meant and can instruct gag at least and/or pol code area and the promotor that is connected with its navigability and the optional assembly of the expression of polyadenylation sequence, described polyadenylation sequence is positioned at the downstream of the nucleotide sequence of encoding gag and/or pol.Described polyadenylation sequence can for example be derived from simian virus 40 (SV40) or bovine growth hormone gene.Many polyadenylation sequences are known in the art.

The packing construct can comprise other code area outside above-mentioned concrete gene.Other gene comprises vif, vpw, vpy, tat and rev gene.In some embodiments, can be from BIV or from obtaining the rev gene as different slow viruss such as HIV.Construct can also comprise the nucleotide corresponding to enough numbers of functional tat gene.

In some embodiments, thus can make as splice site inactivations such as main donor splicing sites or with its elimination and reduce or eliminate aberrant splicing.Can finish sequence variation by standard technique known in the art such as packaging sequence and/or donor splicing site.

Some embodiment of the present invention also provides minimum retrovirus packing construct or slow virus packing construct.In some embodiments, construct comprises the polyadenylation signal of 3 ' end of the promotor that is connected with gag/pol coded sequence navigability and described gag/pol coded sequence.In some embodiments, described packing construct is included in the allos intron of described gag/pol coded sequence upstream (promptly 5 ' locating).In addition, the packing construct can contain the rna transport element.In some embodiments, this element can be slow virus RRE, BIVRRE, or it can be CTE as herein described.In some embodiments, the packing construct can also comprise the Rev coded sequence.In some embodiments, can change the gag/pol code area and not change amino acid sequence, thereby but for example by as with the codon of optimization gag/pol code area recompile has been eliminated needs to RRE.

In some embodiments, virocyte surface protein expression construct of the present invention is to carry the expression construct of coating coded sequence and comprise VSV-G env code area.In some embodiments, because VSV-G gives the host range of recombinant virus broadness, thereby VSV-G albumen is desirable env gene.Yet in some cases, VSV-G env may have harmful effect to host cell.Thereby in some embodiments, when using as during the code areas such as code area of VSV-G env, adopts controlled gene expression mechanism (as the inducible promoter system etc.) thus can regulate so that when not needing the VSV-G expression, make host cell toxicity minimum the VSV-G expression.For example, can adopt Gossen ﹠amp; Bujard (Proc.Natl.Acad.Sci. (1992) 89:5547-5551) but the abduction delivering that VSV-G can be provided by the gene expression system that tetracycline is regulated.In some embodiments, the agent of tet/VP16 trans-activation may reside on first carrier, and the VSV-G coded sequence can be cloned into the downstream of the promotor of the tet operon sequence control that is subjected on another carrier.Other limiting examples of regulatable expression system has been described in open WO 01/30843 of PCT and WO 02/06463.

In some embodiments, the construct of coding coating of the present invention comprises LCMV mutant env code area (Beyer etc., J.Virol., 76:1488-1495,2002), the high soil of holder (Thogoto) coating (for example, see that PCT discloses WO03066810 number) and/or baculoviral gp64env code area (Monsma etc., J.Virol.70 (7): 4607-4616,1996).In one embodiment, LCMV mutant env and/or pg64env code area are expressed by composition ground.In another embodiment, express LCMV mutant env and/or pg64env code area from inducible promoter.Inducible promoter system and constitutive promoter system be known in the art (for example see, WO03066810) and this paper also some of them are described.Other coating can be used for practice of the present invention and include but not limited to 4070A env, muroid leukemia virus (MuLV) env, Moloney muroid leukemia virus env, amphophilic env, different preferendum env, parent's preferendum env, many preferendums env, GP120env from HIV, HTLV I env, HTLV II env, hepatitis B (HBV) env, as influenza env such as HA, rabies virus glycoprotein (GP), Alphavirus GP, Luo Xi river virus GP, Semliki Forest virus (Semliki Forest virus) GP, sindbis virus GP, filamentous form virus GP, Ebola virus (for example, Zaire's bacterial strain) env, Marburg virus env, γ retrovirus GP and EBV env, for example also see, Reiser, Gene Therapy 7:910-913 (2000) and Cronin etc., Curr Gene Ther 5 (4): 387-398 (2005).These coatings are not limited to use in BIV or slow virus carrier, but can or have envelope vector to use with any suitable or compatible envelope virus.

In some embodiments, viral vectors of the present invention comprises decay accelerating factor (DAF).For example, tunicary viral vectors comprises DAF on viromembrane.In some embodiments, DAF is wild type DAF.In some embodiments, DAF is a part that has the fusion of envelope protein, for example, sees Guibinga etc., Mol Ther.200511 (4): 645-51.The present invention comprises that also the BIV that expresses DAF produces cell.

Can be used to transform any basically cell-line or the host cell that can be used as incasing cells or produce cell according to some virus formulation body of the present invention (for example, BIV construct).These cells can be prokaryotic host cell or eukaryotic host cell.Described cell can be bacterium, saccharomycete, insect cell or the like.Transform and undertaken by transfection or transduction usually.Transfection is with transforming target cell or host cell as separated DNA genomes such as plasmids, seeing for example Kriegler, M., Gene Transferand Expression:A Laboratory Manual, W.H.Freman ﹠amp; Company NY (1990).Can be with reference to being purchased kit, as the CalPhos kit (Clontech Inc.Palo Alto, Calif.) and Profection kit (Promega, Madison Wis.) etc.In addition, the example of centrifugal transfection (spinoculation) method is seen Kotani etc., Human Gene Ther.5:19-28 (1994) and Forstell etc., J.Virol.Methods 60:171-178 (1996).In some embodiments, cell is as mammalian cells such as primate cell or human cells.Example includes but not limited to that the human embryonic kidney cell (for example, 293 or 293T), rabbit EREp cell, Cf2Th (ATCC No.CRL 1430), CHO, SW480, CV-1, human T-cell be CEM-SS, Jurkat, MDCK and D17 dog cell-line, HT1090, LINA, WES, and as muroid cell-lines such as NIH3T3.The eukaryotic that cell-line or cell culture are illustrated in growth in vitro or keep.The filial generation that is appreciated that cell may be not exclusively identical with parental cell (for example, on the form, on the genotype or on the phenotype).

Because incasing cells and/or production cell can be the human cells, and the target cell can be the human cell, therefore can select the next expression that optimization is provided of various elements of interested vector construction body in particular host cell and target cell, for example, the optimizing codon of people's origin promoter, personnel selection is to code area recompile etc.

In case with vector construction body of the present invention transfection or transduceed incasing cells and/or production cell, then gene will will be made new virion by expression and described cell.As a result, virion can be collected and be used for infecting or transduction target cell, thus with the interested one or more gene transfer in the described carrier in the target cell.Adopt the transduction method of virus to comprise directly cultivating (Bregni etc., Blood 80:1418-1422 (1992)) altogether or cultivating (Xu etc., Exp.Hemat., 22:223-230 (1994) separately or with suitable growth factor of cell with viral supernatant.In some embodiments, can and/or concentrate viral virion purifying (partial purification or almost completely purifying).

Some embodiment of the present invention also relates to sets up stable package cell line and production cell-line so that preparation virus.Each slow virus proteinase activity site mutation makes can make up the slow virus package carrier, and described slow virus package carrier can be used for setting up the stabilising packaging cell-line that is used for producing slow virus carrier, for example, sees United States Patent (USP) the 7th, 070, No. 993.In brief, the catalytic center of hiv protease comprises triamido acidic group preface Asp-Thr-Gly (for example, seeing Konvalinka, J. etc., J.Virol.69:7180-7186,1995).(Korber B etc., Science (1998) 280:5371) that these three amino acid are normally guarded in the HIV that is put down in writing up to now, BIV, EIAV, FIV and SIV separated strain.Described Thr residue (corresponding to the 26th amino acids of the protease gene section start from HIV separated strain HXB2) is become for example sudden change of Ser produced the continuation that can allow in the host cell, the functional protein enzyme of constitutive expression.

So, in one embodiment, the invention provides the sudden change from Thr to Ser in the Asp-Thr-Gly motif of protease (for example, BIV or hiv protease).Some embodiment of the present invention adopts the stabilising packaging cell-line based on BIV to be used for slow virus carrier production based on BIV, thereby comes BIV gag/pol expression with this point mutant in the described protease-encoding district.Such stabilising packaging cell-line can allow to generate the cell-line of such generation biv lentiviral vector: the virion that can produce high titre.

" corresponding to " sudden change that replaces of T → S in the described encoded slow virus protease can be this T → S replacement itself or in described protease identical or similar motif place have the replacement of biological effect of equal value.Those skilled in the art can easily assess other and replace to check whether they cause biological effect of equal value." biological effect of equal value " is meant that the expression that causes composition and continuation in the host cell replaces, described replacement has kept replacing or the similar virus protein enzyme activity level of wild-type protease to described T → S, Konvalinka etc., J.Virol.69:7180-7186,1995." virus protein enzymic activity " can be as Konvalinka J. etc., and J.Virol.69:7180-7186 measures described in 1995.When described T → S is replaced and this site with another amino acid whose be substituted in the difference measured under the essentially identical experiment condition less than 2 times, less than 1.5 times or even during less than 1.2 times, active is " similar " in implication of the present invention with cytotoxicity.In some embodiments, in the time of in being used in production system, the replacement at identical or similar motif place will produce similar virion output in described protease, for example compare with the production system that adopts corresponding wild-type protease about 3 times of differences with interior, about 4 times of differences with interior, about 5 times of differences with interior, about 2 times of differences with interior, about 1.5 times of differences with interior or about 1.2 times of differences in, for example in the transient transfection system.Described multiple difference may less than and/or greater than output with corresponding wild-type protease.

In some embodiments of the present invention, with the security feature through engineering approaches to as in the slow virus carrier systems such as HIV or BIV.Exploitation can be to produce the cell of described carrier can generate " works " virus (for example, the virus that can breed or can breed in the production extracellular) in the target cell possibility elimination or minimum based on one of main instruction of the carrier system of virus.For this reason, some carrier system of the present invention (security feature that for example, BIV) has added other carrier system (for example, other slow virus carrier) that has equaled or exceeded present utilization.Some features that can add in BIV of the present invention, retrovirus or the lentivirus production system include but not limited to: gag and/or RRE sequence are minimized; Make the gag ATG sudden change in the carrier; The carrier main chain can be from inactivation (SIN); The tat gene can be eliminated from described system; And/or can eliminate all or some episomes.In some embodiments, used with design to minimize, to reduce or to eliminate the carrier system based on virus of the chance that viral vectors can breed or duplicate in the target cell.In some embodiments, used through the carrier system based on virus of design to allow viral vectors in the target cell, to breed, propagate and/or duplicate.This comprises that the controlled of propagation that can limit described virus duplicate, and for example, is restricted to taking turns and duplicate (cause transformant to produce vector particles as initial conversion, described vector particles is " released " and transforms second group of cell) in the target cell.In some embodiments, described second group of cell do not produce vector particles.Duplicating virus carrier or limited duplicating virus carrier may cause by the increase of transformant quantity.

Biv vector system and the application in nucleic acid is carried thereof have and help good drug safety feature.For example, BIV can not cause human diseases; Usually biv vector does not have significant sequence homogeneity with human pathogen HIV; BIV is the virus of becoming estranged most with HIV in the slow virus phylogenetic tree; And HIV does not forward the biv vector bag in the virion to.Thereby HIV infects can not make the movable and propagation of therapeutic biv vector.

Albumen of the present invention

The invention provides the method for expressing and producing albumen of the present invention (for example, antibody as herein described or albumen analog).The present invention also provides the separated nucleic acid of the albumen of the present invention of encoding, the carrier that comprises described nucleic acid and host cell, and the recombinant technique that is used to generate described albumen.

For the generation albumen of recombinating, can be with (for example, replicable vector) in the nucleic acid insertion carrier of this albumen of coding so that further clone (described DNA for example, increases) or so that expression.In some embodiments, the encode sequence of albumen of the present invention can be to comprise the coded sequence that has carried out the codon optimized for the cell of expressing described albumen.In another embodiment, can generate albumen by homologous recombination, for example, as United States Patent (USP) the 5th, 204, No. 244 described.

Can utilize many carriers.Carrier components generally include but be not limited to following one or more: burst, origin of replication, more than one marker gene, enhancer element, promotor, transcription terminator, for example, as United States Patent (USP) the 5th, 534, No. 615 are described.

The proper host cell that is used for cloning with the code area of expression vector is prokaryotic, yeast cell or higher eucaryotic cells.The prokaryotes of suitable this purpose include but not limited to as eubacterias such as gram-negative biological or Gram-positive biologies, for example, as Escherichia (Escherichia) (for example, Escherichia coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) (for example, salmonella typhimurium (Salmonella typhimurium)), Serratia (Serratia) (for example, serratia marcescens (Serratia marcescens)) and Shigella enterobacteriaceaes (Enterobacteriaceae) such as (Shigella), and as bacillus subtilis (B.subtilis) and bacillus licheniformis (B.licheniformis) bacillus (Bacilli) such as (for example, bacillus licheniformis 41P), as pseudomonas aeruginosa pseudomonas (Pseudomonas) and streptomyces (Streptomyces) such as (P.aeruginosa).In some embodiments, the escherichia coli cloning host is that (for example, ATCC 31 for Escherichia coli 294,446), however as Escherichia coli B, Escherichia coli X1776 (for example, ATCC31,537) and Escherichia coli W3110 (for example, ATCC 27,325) wait other bacterial strain also to be fit to.These examples are illustratives and nonrestrictive.

Except that prokaryotes, also be clone or the expressive host that is fit to as eukaryotic microorganisms such as filamentous fungi or saccharomycete.What use always in low microorganism such as eucaryon host such as grade is saccharomyces cerevisiae (Saccharomyces cerevisiae) or common Saccharomyces cerevisiae.Yet, much other genus, kind and bacterial strain also be use always and can be used for herein, as the chestnut wine fission yeast (Schizosaccharomycespombe); Kluyveromyces (Kluyveromyces) host, for example, Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis) (for example, ATCC 12,424), Bulgaria's kluyveromyces (K.bulgaricus) (for example, ATCC 16,045), Wei Shi kluyveromyces (K.wickeramii) (for example, ATCC 24,178), Wa Erte kluyveromyces (K.waltii) (for example, ATCC 56,500), have a liking for and (for example reveal kluyveromyces (K.drosophilarum), ATCC 36,906), heat-resisting kluyveromyces (K.thermotolerans) and kluyveromyces marxianus (K.marxianus); Inferior sieve saccharomyces (yarrowia) (for example, EP402,226); Pichia pastoris (Pichia pastoris) (for example, EP183,070); Candida (Candida); Trichoderma reesei (Trichoderma reesia) (for example, EP244,234); Neurospora crassa (Neurospora crassa); Permitted prosperous yeast (Schwanniomyces occidentalis) etc. as the west and permitted prosperous saccharomyces (Schwanniomyces); And filamentous fungi, for example Neurospora (Neurospora), Penicillium (Penicillium), Dendrochium (Tolypocladium) and as aspergillus nidulans (A.nidulans) and aspergillus niger aspergillus (Aspergillus) hosts such as (A.niger).

The suitable host cell that is used to express glycosylated protein can be derived from multicellular organism.The example of non-vertebrate cells comprises plant cell and insect cell.Multiple baculoviral bacterial strain and variant and come freely fall army worm (Spodoptera frugiperda) (caterpillar), Aedes aegypti (Aedes aegypti) (mosquito), aedes albopictus (Aedes albopictus) (mosquito), Drosophila melanogaster (Drosophila melanogaster) (fruit bat) and silkworm hosts' such as (Bombyx mori) corresponding permission insect host cell to be identified and can be used for expressing protein.The multiple Strain that transfection is used can be used for protein expression and can openly obtain, for example, the L-1 variant of autographa california (Autographa californica) NPV and the Bm-5 strain of silkworm NPV, and these viruses can for example be used for the transfection of fall army worm cell at this as according to virus of the present invention.The plant cell cultures of cotton, cereal, potato, soybean, petunia, tomato and tobacco also can be used as the host.

Some embodiment of the present invention utilizes vertebrate cells, and the propagation of vertebrate cells in culture (tissue culture) can be conventional method.The example of useful mammalian host cell line is the monkey kidney CVI system (for example, COS-7, ATCC CRL 1651) by the SV40 transfection; Human embryonic kidney cell system (for example, thus comprise 293 or 293T cell of 293 or the 293T cell-line that can in suspension culture, grow through subclone, Graham etc., J.Gen Virol.36:59 (1977), as 293 Freestyle (Invitrogen, Carlsbad, CA) etc.) or 293FT; Baby hamster kidney cell (for example, BHK, ATCC CCL 10); Chinese hamster ovary cell; Chinese hamster ovary cell/-DHFR (for example, CHO, Urlaub etc., Proc.Natl.Acad.Sci.USA 77:4216 (1980)); Mouse supportint cell (for example, TM4, Mather, Biol.Reprod.23:243-251 (1980)); MK cells (for example, CVI ATCC CCL 70); African green monkey kidney cell (for example, VERO-76, ATCCCRL-1587); Human hela cell (for example, HELA, ATCC CCL 2); MDCK (for example, MOCK, ATCC CCL 34); The CF2TH cell; Buffalo rat hepatocytes (for example, BRL 3A, ATCC CRL 1442); Human pneumonocyte (for example, W138, ATCC CCL 75); Human hepatocytes (for example, Hep G2, HB 8065); Mouse mammary tumor (for example, MMT 060562, ATCC CCL51); TRI cell (Mather etc., Annals N.Y.Acad.Sci.383:44-68 (1983)); MRC 5 cells; The FS4 cell; With human liver cancer cell-line (Hep G2).

Usually expression vector of using with production albumen or cloning vector come transformed host cell and it are cultivated in conventional nutrient medium, are applicable to the gene of evoked promoter, selection transformant, the required sequence of amplification coding or are applicable to downstream purification and/or concentration step thereby described conventional nutrient medium is modified.

In some cases, host cell can be modified so that reduce or eliminate the expression of endogenous protein.For example, if (for example, Chinese hamster ovary celI) generates the factor B analog in particular host cell, thereby then can modify the expression that described host cell reduces or eliminate the natural factor B (for example, hamster factor B) of described host cell.Therefore, the invention provides the method that generates the complement protein analog, described method comprises the expression that reduces or eliminate natural complement protein corresponding in the host cell.The method that is used to reduce, eliminate or knocks out the expression of host cell proteins is known in the art.For example, can be by having specific inhibitory RNA (for example, RNAi) to reduce or eliminate the expression of described albumen with the RNA that expresses encoding proteins the host cell through engineering approaches.For example, (Mountain View CA) sells and to be used for striking various carriers and the kit that subtracts the host cell protein expression Clontech, as KNOCKOUT^TMThe carrier of the part of RNAi system and kit.Other method comprises the gene targeting by homologous recombination, and this makes can introduce specific sudden change in any cloned genes, for example, see Current Protocols in Molecular Biology, John Wiley; Sons, Inc., 1994-1998, the 9.16th joint and the 9.17th joint.This can be used to knock out the gene of expression host cell albumen.

The another kind of method that can be used for reducing the expression of endogenous protein relates to uses the directed transcription factor of preventing endogenous protein to be expressed.For example, can preventing the territory to connect or be fused to as DNA such as zinc finger polypeptides with transcription factor in conjunction with the territory.Those skilled in the art can design the zinc finger polypeptide that combines with specific dna sequence, for example, see United States Patent (USP) the 6th, 140, and No. 081 and the 7th, 067, No. 617; And U.S.'s publication application No. 20060078880, No. 20040224385 and No. 20070213269.Those skilled in the art can be relevant with zinc finger polypeptide and transcription repression territory (for example, KRAB (the Kr ü ppel associated cartridge) territory) that designs.(Proc Natl Acad Sci USA.200097 (4): 1495-1500) and described the example of these molecules and technology in No. 20070020627, U.S.'s publication application at Beerli etc.In some embodiments of the present invention, can use the carrier transduction host cell of expressing transcription repressor.This method knocks out interested gene than with homologous recombination and more has superiority, and this is because host cell is dliploid and it is desirable to two gene copies are all knocked out in most of situations.Yet the expression of transcription repressor can be prevented the expression of two gene copies.

The compound that also can the employing meeting directly or indirectly reduces the expression of specific endogenous protein reduces the expression of described specific endogenous protein.With fB is example, can use all cpds to reduce the expression of endogenous fB representation.For example, suppress having of fB expression according to the show: histamine (Falus﹠amp; Meretey, Immunology 1987 60:547-551 and Falus ﹠amp; Meretey, Mol Immunol198825 (11): 1093-97), sodium butyrate (Andoh etc., Clin Exp Immuno 1999118:23-29), as glucocorticoids such as dexamethasone (Dauchel etc., Eur J Immunol 199020 (8): 1669-75), platelet derived growth factor (Circolo etc., 1990 The Journal of BiolChem 265 (9): 5066-5071), epidermal growth factor (Circolo etc., 1990) and fibroblast growth factor (Circolo etc., 1990).Thereby can in the presence of any of these molecules or combination, cultivate the endogenous expression that host cell of the present invention reduces fB and possible fD.This is used in and produces fB analog or fD analog in the host cell.Therefore, In some embodiments of the present invention, under the existence of any or multiple compound that is selected from the group of forming by histamine, sodium butyrate, glucocorticoid (for example, dexamethasone), platelet derived growth factor, epidermal growth factor or fibroblast growth factor, the host cell of expressing fB analog or fD analog is cultivated.

All cpds and albumen can raise or keep the expression of fB according to the show.For example, the fB representation is raised or keeps by following material according to the show: TNF (Andoh etc., Clin Exp Immuno 1999118:23-29), oestrogenic hormone (Sheng-Hsiang etc., Biology of Reproduction 200266:322-332), il-1 (Dauchel etc., Eur J Immunol 199020 (8): 1669-75), dexamethasone (Lappin ﹠amp; Whaley, Biochem J 1991 280:117-123), prednisolone (Lappin ﹠amp; Whaley 1991), cortex (cortical) (Lappin ﹠amp; Whaley 1991) and gamma interferon (Huang etc., 2001 Eur J Immunol 31:3676-3686).Thereby can not exist any of these molecules or combination the time cultivate the endogenous expression that host cell of the present invention reduces fB and possible fD.In addition, can in the presence of the inhibitor of any one or more these compounds, cultivate host cell.This is used in and produces fB analog or fD analog in the host cell.Therefore, In some embodiments of the present invention, the host cell of culture expression fB analog or fD analog in the presence of any or multiple compound of the compound that can suppress to be selected from the group of forming by TNF, oestrogenic hormone, il-1, dexamethasone, prednisolone, cortex and gamma interferon.In some embodiments, for example use method as herein described to reduce of the expression of one or more these compounds by host cell.The example of estrogenic inhibitor includes but not limited to tamoxifen.Inhibitor also comprises and combines with described compound and reduce its active antibody.For example, it is known in the art combining and make the various antibody of its inactivation with TNF.

The host cell that is used for producing albumen of the present invention can be cultivated at various medium.As Ham ' s F10 (for example, Sigma), MEM ((MEM), for example, Sigma), RPM 1-1640 (for example, Sigma) and the improved Eagle medium of Dulbecco ((DMEM) for example, Sigma) waits and to be purchased medium and to go for cultivating described host cell.In addition, at Ham etc., Meth.Enz.58:44 (1979); Barnes etc., Anal.Biochem.102:255 (1980); United States Patent (USP) the 4th, 767, No. 704, the 4th, 657, No. 866, the 4th, 927, No. 762, the 4th, 560, No. 655 or the 5th, 122, No. 469; WO 90/03430; WO 87/00195; Or United States Patent (USP) issues that again any medium of describing in the 30th, No. 985 also can be used as the medium of described host cell.In some embodiments, medium can be completely specified (for example, CD-CHO medium (Invitrogen)), serum-free or contain serum.Any of these medium can be supplemented with where necessary: hormone and/or other growth factor (as insulin, transferrins or epidermal growth factor etc.), salt (as sodium chloride, calcium salt, magnesium salts and phosphate etc.), buffer (as HEPES etc.), nucleotide (as adenosine and thymidine etc.), antibiotic are (as GENTAMYCIN^TMMedicine etc.), the energy source of trace element (being defined as the inorganic compound that exists with the ultimate density in the micro-molar range usually) and glucose or equivalence.Can also comprise coming any other necessary fill-in with debita spissitudo well known by persons skilled in the art.As condition of culture such as temperature and pH values is the used condition of before this selection being expressed of host cell, or in those skilled in the art's development technique.

When using recombinant technique, can make albumen be created in the cell, in periplasmic space and/or direct secretion in medium.In some embodiments, if albumen is created in the cell, at first remove no matter host cell still is the particulate debris of cleaved fragment by for example centrifugal or ultrafiltration.The Bio/Technology 10:163-167 (1992) of Carter etc. has described the method that is used to separate the albumen (antibody) that is secreted into colibacillary periplasmic space.In some embodiments, albumen is secreted in the medium.In some embodiments, usually at first will be from the supernatant of these expression systems, for example for example Amicon or Millipore Pellicon ultra filtration unit concentrate with being purchased the thickening filtration device.In some embodiments, can in any above-mentioned steps, comprise as protease inhibitors such as PMSF so that Profilin hydrolysis and/or comprise that into antibiotic is so that prevent the growth of exogenous impurity.

Can use that for example hydroxylapatite chromatography, gel electrophoresis, dialysis, size exclusion chromatography, affinity chromatography, immune affinity chromatographic, tangential flow purification, diafiltration, ion-exchange chromatography, reverse-phase chromatography, heparin sepharose affinity chromatography and other separation known and conc forms are carried out purifying to the protein composition from cell preparation.

When described albumen is antibody, can utilize albumin A to come purifying.Albumin A depends on the kind and the isotype in any immunoglobulin Fc territory that exists in the described antibody as the suitability of affinity ligand.Albumin A can be used for the antibody (Lindmark etc., J.Immunol.Meth.62:1-12 (1983)) of purifying based on people γ 1,γ 2 orγ 4 heavy chains.Recommend to adopt Protein G (Guss etc., EMBO are (1986) J.5:1567-1575) for all mouse isotypes with to people γ 3.In some embodiments, the matrix that is connected with affinity ligand is agarose, but also can utilize other matrix.As mechanically stable matrix such as controlled cellular glass or poly-(styrene divinyl) benzene can allow to compare the usefulness agarose obtainable flow velocity faster and shorter processing time.When described antibody contains C_HDuring 3 territories, BAKERBOND ABX^TM(J.T.Baker, Phillipsburg NJ) can be used for purifying to resin.Also can utilize other protein purification technology according to albumen to be recycled or antibody, as ion exchange column separation, precipitation with alcohol, reversed-phase HPLC, silica gel chromatograph, heparin SEPHAROSE^TMChromatogram, anion or cation exchange resin chromatography (as poly-aspartate post etc.), SDS-PAGE and ammonium sulfate precipitation etc.

After any preliminary purification step, the mixture that comprises described protein of interest and any possible impurity is hanged down pH value hydrophobic interaction chromatograph, for example, use the pH value to be about 2.5～4.5 elution buffer, in some cases under low salt concn (for example, the salt of 0～0.25M) carry out or other the operation to be further purified.

Eye is carried or therapy

The selection that the eye gene therapy is used is relevant with the safety of described therapy.Carrier obtains continuous expression by its transgenosis Payload is incorporated in the target cell genomic dna usually.The integration incident may cause the local failure that is called " inserting sudden change " in the target cell DNA.In some embodiments, biv vector is the targeted cells type specifically, for example, and the RPE cell.The RPE cell does not divide usually; The RPE cell seldom causes tumour; The a limited number of RPE cell of only need transduceing in some cases; And the RPE cell of transduction is confined to the injection site place with maintenance, and this can pass through noninvasive method (for example, ophthalmoscopy) in some cases and repeatedly observe.In some embodiments, the noninvasive method of observation usefulness is ophthalmoscopy and can eliminates RPE cell (the RPE cell of for example, having transduceed) by laser therapy.Thereby, be specially adapted to genetic modification as the RPE cell of the target cell that is used to produce product (for example, as therapeutic products such as albumen or siRNA).At last, thus can carry out through engineering approaches to carrier and realize site-directed integration.A kind of mode that obtains such directional integration is by using the specific nucleic acid binding molecule, as contains the molecule of zinc-finger motif (for example, zinc-finger motif) relevant with integrase.Another mode of tackling this problem is to use the carrier that dissociates as maintenances largely such as AAV.

Fig. 1 is the example of AMD progress.The feature of early stage AMD is drusen normally, a kind of sediments between retinal pigment epithelium (RPE) layer and Bruch's membrane.Drusen can play blocking-up nutriment, oxygen and/or refuse retina with below the CC bed between mobile effect.Importantly, drusen contains many inflammatory factors and complement factor.The course of disease of AMD is mainly carried out at one of both direction.In moist AMD, new blood vessel grows outside the choroid by the process that is called angiogenesis.Described neovascularity is defective.Their seepages are also hemorrhage, and this causes rapidly losing one's sight.Second kind of course of disease is ground pattern atrophy.In this case, thus retina, RPE layer and choroid are all dead to cause not having zone, amphiblestroid eyeground to enlarge.The zone of described expansion is relevant with blind spot, and this finally can eliminate central vision and cause losing one's sight.Importantly, some embodiment of the present invention can be treated all stages of AMD; That is the AMD in late period of early stage AMD and two kinds of forms.The present invention can be as the general treatment of AMD.

Composition, preparation and goods

Some embodiment of the present invention provides as being used for the treatment of the composition of purposes, for example, and pharmaceutical composition.In some embodiments, composition comprises complement analog as described herein.In some embodiments, the external activity of the external activity by more naturally occurring albumen and the external activity of described analog and/or more naturally occurring albumen and the activity in vivo of naturally occurring albumen are (for example, in animal model), calculate or release the activity in vivo of the expection and/or the expectation of described analog then, and in some cases any difference of half life is adjusted, thereby determine " treatment effective dose " or suitable dosage.

Preparation (for example, injection preparation) usually but may not be the biocompatible solution of active component for example, comprises Hanks liquid or Ringer's solution.The preparation of transdermal administration or saturating mucosal administration generally includes but is not limited to bleeding agent such as fudidic acid or bile salt etc. with washing agent or combinations-of surfactants.In some embodiments, preparation can be made as aerosol, suppository or patch.In some embodiments, unfavorable for albumen or the Orally administered possibility of peptide active component; Yet, may be fit to this based composition for example is formulated in enteric coating form, storage agent, capsule or the like, so that protect it to avoid the digestive ferment influence, thereby also can adopt Orally administered.Some preparation of the present invention comprise balanced salt solution (Alcon Laboratories, Inc., Fort Worth, Texas) or balanced salt solution plus (Alcon Laboratories, Inc.).In some embodiments, preparation comprises more than one following material: citrate, NaCl (for example, 0.64%), potassium chloride (KCl) (for example, 0.075%), two hydration calcium chloride (CaCl₂2H₂O) (for example, 0.048%), Magnesium dichloride hexahydrate (MgCl₂6H₂O) (for example, 0.03%), sodium acetate trihydrate (CH₃CO₂Na3H₂O) (for example, 0.39%), two hydration sodium citrate (C₆H₅O₇Na₃2H₂O) (for example, 0.17%) and sodium hydroxide and/or hydrochloric acid (in order to regulate the pH value) and water.Previous list comprises some molecule of classifying specific hydrate (for example, dihydrate, trihydrate, hexahydrate etc.) as.Be appreciated that the various hydrates that can use these compounds in the present invention and the invention is not restricted to these specific hydrated forms of listed molecule.In some embodiments, preparation comprises hydrochloric acid and/or the sodium hydroxide and the water of more than one following material: NaCl, a hypophosphite monohydrate dihydric salt, seven hypophosphite monohydrate disodium hydrogens and adjusting pH value.In some embodiments, preparation comprises more than one following material: histidine (for example, about 10mM), α, α-dehydration trehalose (for example, about 10% or about 50mM), MgCl₂(for example, about 10mM), as polysorbate (for example, about 0.01%) and NaCl such as polysorbates 20₂(for example, about 0.1%).In some embodiments, preparation comprises molecule of the present invention, 10mM histidine, 10mMMgCl₂, 50mM trehalose and 0.01% polysorbate 20 or form by them.In some embodiments, preparation comprises molecule of the present invention, 1.0%NaCl and 10mM MgCl₂Or form by them.In some embodiments, the pH value of preparation or composition is about 5.5.In some embodiments, the pH value of preparation or composition is about 5.0～9.0, about 5.0～5.5, about 5.3～5.7, about 5.5～6.0, about 5.8～6.2, about 6.0～6.5, about 6.3～6.7, about 6.5～7.0, about 6.8～7.2, about 7.0～7.5, about 7.3～7.7, about 7.5～8.0, about 7.8～8.2, about 8.0～8.5, about 8.3～8.7 and about 8.5～9.0, any value that is suitable for keeping the stability of biologically active and active component.

The suitable preparation of the mode of administration that is used to expect and the example of compound method are found in No. the 7th, 208,577, " Remington ' s Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA " and the United States Patent (USP).Dosage level and accurate prescription also can be determined by optimization routine method well known in the art.

Some embodiment of the present invention provides the pharmaceutical composition of complement analog (for example, factor B analog).In some embodiments, thus the activity in vivo of external activity by the external activity of for example more naturally occurring albumen and the external activity of described analog, more naturally occurring albumen and naturally occurring albumen, calculate the expection activity in vivo of described analog and the difference of any mensuration of half life adjusted determine treatment effective dose or suitable dosage then.In some embodiments, according to determining the treatment effective dose before this as clinical testing etc.In some embodiments, by changing as the dosage among the patient until obtaining desired effects or realizing treating benefit and determine the treatment effective dose.Guide to concrete dosage and carrying method is provided in the document; For example see United States Patent (USP) the 4th, 657, No. 760, the 5th, 206, No. 344 or the 5th, 225, No. 212.

In some embodiments, the composition of using in the body contains " carrier " or " the acceptable carrier of medicine ".Term " carrier " is meant thinner, adjuvant, the excipient of using with interested carrier or carries device.Term " carrier " includes but not limited to solid material or fluent material, described solid material or fluent material can be inorganic matter or organic matter and can be derived from syntheticly or natural, described solid material or fluent material are mixed with the active component of composition or prepare so that promote using study subject.Any other material that custom adopts in compounding pharmaceutical may be suitable.The solid carrier includes but not limited to natural and synthetic cloisonne enamel silicate, for example, and as natural silicates such as diatomite; Magnesium silicate, for example, talcum; Aluminium-magnesium silicate, for example, Attagel and vermiculite; Alumina silicate, for example, kaolinite, montmorillonite and mica; Calcium carbonate; Calcium sulphate; Synthetic hydration silicone oxide and synthetic calcium silicates or alumina silicate; As elements such as carbon or sulphur; As natural resin or synthetic resin such as polyvinyl alcohol; And as waxes such as paraffin and beeswaxs.The example of the liquid carrier that is fit to comprises water; Contain the aqueous solution just like oxygen-containing organic compounds such as ethanol and oils, described oils comprises and is derived from oil, animal, plant or synthetic oils, as peanut oil (for example, low irritated peanut oil), soybean oil, mineral oil and sesame wet goods.In some embodiments, carrier can be water, physiological saline, dextrose solution and glycerite or buffer solution.In some embodiments, composition comprises more than one the acceptable carrier of medicine, as the salt solution of salt solution, phosphate-buffered and/or controlled release preparation etc.Also can exist as flavor enhancement, fumet, colouring agent and suspending agent etc. and be present in buffer solution and other material in the pharmaceutical preparation usually.The medicine carrier can be different from typical solution and suspension, its difference is that the medicine carrier got rid of by special preparation and/or minimize the amount of substance that may be harmful to the host of its applying said compositions or availability (for example, removing bacteriotoxin) so that use in vivo.

Usually, water, suitable oil, salt solution, dextrose (glucose) aqueous solution and relevant sugar juice and be the carrier that is suitable for the outer solution of stomach and intestine usually as di-alcoholss such as propane diols or polyethylene glycol.In some embodiments, be used for water soluble salt that solution that stomach and intestine use contains active component, suitable stabilizing agent and if desired or buffer substance in case of necessity outward.Independent or what mix is suitable stabilizers as antioxidants such as sodium hydrogensulfite, sodium sulphite or ascorbic acid.Also adopt citric acid and salt thereof and EDTA sodium in addition.In addition, the outer solution of stomach and intestine can contain Benzalkonii Chloridum, methyl p-hydroxybenzoate or preservatives such as propylparaben and chlorobutanol.

Suitable drug excipient includes but not limited to starch, glucose, lactose, sucrose, gel, antibiotic, preservative, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, odium stearate, glycerin monostearate, talcum, sodium chloride, skim milk powder, glycerine, propylene, glycol, water and ethanol or the like.Composition also can contain wetting agent and/or emulsifier and/or pH buffer when needed.Where necessary, the composition local anesthetic that also can comprise solubilizer and/or ease the pain at the injection site place as lidocaine etc.

In the canonical reference book in this area " Remington ' s Pharmaceutical Sciences, Mack Publishing Co. " suitable medicine carrier has been described.Some embodiment of the present invention comprisesuse 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or carrier more than 15 kind and/or excipient.

Also active component can be enclosed in the microcapsules that for example pass through condensation technique or interfacial polymerization preparation, for example, CMC in colloid drug delivery system (for example, liposome, albumin microsphere spheroid, microemulsion, nano particle and Nano capsule) or huge emulsion or gelatin microcapsules and poly-(methyl methacrylate) microcapsules respectively.These technology are disclosed in " Remington ' sPharmaceutical Sciences, Mack Publishing Co. ".

Can use stabilizing agent in the present invention.Stabilizing agent is meant the excipient that a class is wide in range, and the envelop of function of this class excipient can be from the swelling agent to the pharmaceutical dissolution or helped prevent sex change or to the additive of the adhesion of chamber wall.Typical stabilizing agent can be the polyhydroxy sugar alcohol; Amino acid is as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine etc.; Organic sugar or sugar alcohol as lactose, trehalose, stachyose, arabitol, antierythrite, mannitol, sorbierite, xylitol, ribitol, inositol (myoinisitol), dulcitol and glycerine etc., comprise as ring-type glycitols such as inositols; Polyethylene glycol; Amino acid polymer; The sulfur-bearing reductant is as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-single thioglycerol and sodium thiosulfate; Low molecular weight polypeptide (for example,＜10 residue); Albumen is as human serum albumins, bovine serum albumin(BSA), gelatin or immunoglobulin; Hydrophilic polymer is as PVP(polyvinyl pyrrolidone), carbohydrate, as monose such as wood sugar, mannose, fructose, glucose or the like; As disaccharides such as lactose, maltose and sucrose; As trisaccharides such as raffinoses; As polysaccharide such as glucan or the like.Usually with every portion of activating agent (for example, as fB analog such as fb3 or in conjunction with antibody or its fragment of fB) 0.1w/w～10, the scope of 000w/w exists stabilizing agent.

Can in the configuration of the therapeutic agent that can be configured to solid dosage forms, comprise disintegrant.Material as disintegrant includes but not limited to starch, and described starch comprises the commercially available disintegrant Explotab based on starch.Sodium starch glycolate, Amberlite (Amberlite), sodium carboxymethylcellulose, over-expense chain starch, sodium alginate, gelatin, orange peel, carboxymethyl cellulose, natural sponge and bentonite can use.The disintegrant of another kind of form is insoluble cationic ion-exchange resin.Powdery natural gum can be used as disintegrant and adhesive, and these powdery natural gum comprise as powdery natural gum such as agar, karaya (Karaya) or bassora gums.Alginic acid and sodium salt thereof also can be used as disintegrant.

Can medicament be reunited with the formation tablet with adhesive, and described adhesive comprises coming the material of natural productss such as gum Arabic, bassora gum, starch and gelatin freely.Other adhesive comprises methylcellulose (MC), ethyl cellulose (EC), carboxymethyl cellulose (CMC) and other cellulose.PVP(polyvinyl pyrrolidone) (PVP) and hydroxypropyl methylcellulose (HPMC) can be used in the alcoholic solution so that described therapeutic agent granulating.

Thereby can in the preparation of therapeutic agent, comprise anti-friction agent and prevent adhesion in process for preparation.Lubricant can be added in the preparation as the layer between therapeutic agent and the die wall and/or with it, these lubricants include but not limited to: stearic acid (comprising its magnesium salts and calcium salt), polytetrafluoroethylene (PTFE), atoleine, vegetable oil and wax.Also can use soluble lubricant, as lauryl sodium sulfate, Stepanol MG, various molecular weight polyethylene glycol (for example, Carbowax 4000 and Carbowax 6000) or the like.

Can add glidant, glidant can improve the flowing property of medicament during preparing and help during pressing rearranges.Glidant can comprise starch, talcum, pyrogenic silica and hydrated aluminosilicate.

In order to help that medicament is dissolved in the aqueous environments, can add surfactant as wetting agent.Surfactant can comprise as anionic detergents such as lauryl sodium sulfate, sodium sulfosuccinate dioctyl ester and dioctyl sodium sulfonates.Can use the cationic detegent that comprises Benzalkonii Chloridum or benzethonium chloride.The nonionic detergent that can comprise in preparation as surfactant includes but not limited to Lauromacrogol 400, stearic acid 40 poly-alkynyloxy group esters,Crodaret 10,50 and 60, glycerin monostearate, polysorbate 20,40,60,65 and 80, sucrose fatty ester, methylcellulose, carboxymethyl cellulose and (for example as any pluronic (pluronic) washing agent such as Pluronic F68 and/or Pluronic F127, see Strappe etc., European Journal ofPharmaceutics and Biopharmaceutics 61:126-133 (2005)).Surfactant can be separately or is present in the preparation of albumen or derivative as the mixture of different proportion.

The additive that might promote the picked-up of albumen (or derivative) includes but not limited to fatty acid, oleic acid, linoleic acid plus linolenic acid.

In some embodiments, controlled release preparation may be desirable.Medicament (for example, albumen) can be added in the inert base that allows to discharge by diffusion, swelling or leaching (for example, natural gum and cellulosic cpd).Also can in preparation, add the matrix of slowly degenerating.Another kind of controlled release forms is by the method based on Oros therapy system (Alza Corp.), and for example, in semipermeable membrane, described semipermeable membrane allows water to enter and make medicine because osmotic effect is extruded by single little opening with drug blockage.Enteric coating has the effect that delays to discharge, but also can have the effect that continues release.

Other dressing also can be used for described preparation.These dressings comprise the various carbohydrates that can be applied in the coating pan.Also can be with medicament with the administration of film garment piece, and the material that is used for this situation can be divided into two groups.First group is non-enteric material and comprises methylcellulose, ethyl cellulose, hydroxyethylcellulose, methyl hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, polyvidone (providone) and polyethylene glycol.Form by the enteric material that is generally phthalic acid ester for second group.

Can use composite material so that the best film dressing to be provided.Can in disc type dressing machine or in fluid bed or by the compression dressing, carry out the film dressing.

In some embodiments, can be neutral form or salt form with nucleic acid of the present invention and particle formulation.Drug acceptable salt includes but not limited to use the salt that forms such as the anion that is derived from hydrochloric acid, phosphoric acid, acetate, citric acid, oxalic acid and/or tartaric acid etc., and the salt that forms such as the cation that is derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, slaked lime, iron hydroxide, isopropylamine, triethylamine, 2-ethyl amido alcohol, histidine, procaine etc.Other stabilizing agent comprises as albumen such as albumin, as emulsifier such as Pluronics, Tweens or the like.In addition, as known in the art, can comprise amino acid, as carbohydrates such as trehalose, mannose, sucrose with have other compound of stabilisation character.

This paper has also considered the pulmonary delivery of medicament of the present invention or albumen (or derivatives thereof).Albumen (derivative) is transported to mammal lung when sucking, and in some embodiments, described albumen (derivative) passes through the lung epithelial layer and enters blood flow and (for example, see Adjei etc., PharmaceuticalResearch 7:565-569 (1990); Adjei etc., International Journal of Pharmaceutics63:135-144 (1990); Braquet etc., Journal of Cardiovascular Pharmacology13 (suppl.5): s.143-146 (1989); Hubbard etc., Annals of Internal Medicine3:206-212 (1989); Smith etc., J.Clin.Invest.84:1145-1146 (1989); Oswein etc., Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colo., March, 1990; Debs etc., The Journal of Immunology 140:3482-3488 (1988); With Platz etc., United States Patent (USP) the 5th, 284, No. 656).

Consider to use the mechanical device of the pulmonary delivery that is designed for the treatment product on a large scale in practice of the present invention, described mechanical device includes but not limited to atomizer, metered dose inhaler and powder inhalator.

Some instantiation that is purchased device that is suitable for the practice of some embodiment of the present invention is by Mallinckrodt, Inc., St.Louis, the Ultravent atomizer that Mo makes; By MarquestMedical Products, Englewood, the Acorn II atomizer that Colo. makes; By Glaxo Inc., Research Triangle Park, the Ventolin metered dose inhaler that N.C. makes; With by Fisons Corp., the Spinhaler powder inhalator that Bedford, Mass. make.

In some embodiments, these devices can use the preparation that is suitable for the albumen dispensing.Usually, each preparation is at used type of device, and except that treatment available thinner, adjuvant and/or carrier, also may relate to the use of suitable propellant material.

In some embodiments, be particle form with protein Preparation.In some embodiments, the average grain diameter of this particle form most preferably is 0.5 μ m～5 μ m, so that be transported to the end of lung less than 10 μ m (or micron).

Carrier can comprise as carbohydrates such as trehalose, mannitol, glutathione, xylitol, sucrose, lactose and sorbierites.Other composition that is used in the preparation for example comprises, DPPC, DOPE, DSPC and DOPC.Can use natural or synthetic surfactant.Can use polyethylene glycol (even except its use in derivatization albumen).Can use as glucans such as cyclisation glucans.In some embodiments, can use cyclodextrin, tertiary amine and/or beta-schardinger dextrin-.Can use the relevant hardening agent of bile salt with other.Can use cellulose and cellulose derivatives.Can use amino acid, as be used in the buffer preparation.Consider to use the carrier of liposome, microcapsules or microsphere, inclusion complex or other type in addition.

In some embodiments, the preparation that is fit to use with atomizer (for example, blast atomizer or ultrasonic atomizer) can comprise in some embodiments the concentration albumen soluble in water with the about 0.1mg of every mL solution～about 25mg biological activity protein usually.Preparation also can comprise buffer solution and/or monose (for example, being used for stabilize proteins and adjusting osmotic pressure).The atomizer preparation also can contain surfactant so that reduce or prevent the spatial induction protein aggregation that solution atomization is caused when forming aerosol.

The preparation that uses with the metered dose inhaler device can comprise divided powder usually, and described divided powder for example contains of the present invention albumen or the medicament of help low suspension in propellant at surfactant.Propellant can be any conventional material that is used for this purpose, as chlorofluorocarbon, HCFC, hydrogen fluorohydrocarbon or hydrocarbon, comprisesArcton 11, dicholorodifluoromethane, dichloro-tetrafluoro ethanol and 1,1,1,2-HFC-134a or its combination.Suitable surfactant comprises sorbitol olein and soybean lecithin.Oleic acid is useful as surfactants also.

In some embodiments, be used for to comprise segmentation dry powder from the preparation of powder inhalator device dispensing, described segmentation dry powder contains medicament of the present invention or albumen and can comprise as swelling agents such as lactose, sorbierite, sucrose, mannitol, trehalose or xylitols, the amount of described swelling agent helps powder is disseminated from described device, for example, 50 weight % of described preparation～90 weight %.

Also considered the nose conveying of albumen.In some embodiments, nose is carried and for example have been allowed that described albumen directly enters blood flow after to the nasal administration medicament.In some embodiments, this described medicament needn't be deposited in lung or the situation of minimal deposition under finish.Nose is carried and is comprised those preparations that have glucan or cyclisation glucan with preparation.Also considered to carry by penetrating transporting of other mucous membrane.

In some embodiments, can be as the preparation of medicaments such as albumen in about 0.10 μ g/kg/ day～produce 10mg/kg/ day desired effects (for example, result of treatment).This paper has described application process and approach.Composition of the present invention and preparation can be used animal by for example infusion (for example, slow infusion) or group's notes.Can maybe itself and other bioactivator together can be used with molecule (s) of interest or interested carrier by infusion (for example, slow infusion) or group annotates, absorb and use by seeing through epithelial layer or mucocutaneous layer.Use and to be systemic application (for example, intravenous is used) or local application.

In some embodiments, use by ocular injection and undertaken.This paper has described various types of ocular injections.In some embodiments, the ocular injection of albumen of the present invention is the about 0.05mg of per injection～about 10mg, about 0.1mg～about 10mg, about 0.5mg～about 10mg, about 1mg～about 10mg, about 5mg～about 10mg, about 0.05mg～about 5mg, about 0.5mg～about 3mg, about 0.5mg～about 1mg, about 0.05mg～about 0.5mg, about 0.05mg～about 0.1mg, about 0.1mg～about 5mg, about 1mg～about 5mg, about 1mg～about 3mg and the described albumen of about 0.5～about 2mg.In some embodiments, carried out the ocular injection of about 5ul～about 150ul, about 25ul～about 150ul, about 50ul～about 150ul, about 100ul～about 150ul, about 5ul～about 100ul, about 50ul～about 150ul, about 25ul～about 150ul, about 25ul～about 100ul or about 35ul～about 70ul.In some embodiments, carried out the ocular injection of about 50ul.

Some preparation of the present invention can also be made as aerosol, suppository, eye drops or patch.

In some embodiments, with composition separately or mix in unit dosage forms and supply with, for example, as being in as the freeze-dried powder in the airtight containers such as ampoule or anther sac or not having aqueous concentrate, described airtight container has been indicated the amount of activating agent usually.When pharmaceutical composition is used by injection, the Injectable sterile water or the salt solution of an ampoule can be provided, thereby described composition is mixed.Composition also can frozen form or liquid form supply.Can comprise stabilizing active ingredient into and/or improve the various compositions (for example, inert fraction) of storage period, for example, as known in the art.

In order to prolong some composition of the present invention (for example, fb3 or antibody) serum circulation in vivo, can adopt various technology.For example, can with or without the locus specificity coupling of multifunction conjunction by polyethylene glycol (PEG) (for example, with the N end of antibody or the terminal coupling of C) or be connected (for example, being connected to antibody) as inert polymer molecules such as high molecular weight PEGs via the ε amino that exists on the lysine residue.In some embodiments, can adopt the linear polymer derivatization or the branch polymer derivatization of the loss that causes the biologically active minimum.Can monitor the degree of coupling closely so that guarantee the PEG molecule and the appropriate coupling of described composition (for example, as albumen such as antibody or fB analogs) by SDS-PAGE and mass spectrum.In some embodiments, can unreacted PEG be separated with the PEG conjugate by size exclusion chromatography and/or ion-exchange chromatography.Adopt method known to those skilled in the art to test to usefulness in the activity of the composition of PEG derivatization and body.

Manufacture

In some embodiments, manufacture contains the material that for example can be used for treating illness as herein described or disease.In some embodiments, manufacture comprises container and label.Suitable containers for example comprises, bottle, bottle, syringe and test tube.Container can be by forming as various materials such as glass or plastics.Container can hold for example can effectively sanatory composition of the present invention.Container can have import and export (as aseptic mouthful etc.), and for example, container can be that have can be by the venous transfusion bag or the venous transfusion bottle of the plug of hypodermic injection needle-penetration.In some embodiments, container can have import and export, for example, but thereby has as pierceable material and/or pliable material etc. and can be penetrated the sealing key element that inclusion is removed.Activating agent in the described composition can be any those activating agents as herein described.In some embodiments, on the container or the label related with container indicate described composition to be used for the treatment of elite illness.In some embodiments, manufacture can comprise second container, and the medicines such as salt solution, Ringer's solution and/or dextrose solution that this second container comprises as phosphate-buffered can be accepted buffer solution.In some embodiments, manufacture comprises the container that fills water.Further can also be included in other material of commercial standpoint and user's position coideal, these materials comprise the package insert of other buffer solution, thinner, filter, syringe needle, syringe and band operation instruction.

Transgenic animal

Some embodiment of the present invention provides the variant of expressing at least a complement pathway composition as herein described or the transgenic animal (for example, non-human animal) of mutant.The method that is used to prepare transgenic animal is known in the art.Some embodiment of the present invention provides the transgenic animal of expressing nucleic acid of the present invention and/or albumen (for example, as factor B analogs such as fB3).In some embodiments, transgenic animal (as mouse etc.) also will be included in sudden change, disappearance or destruction in the Fas gene, for example, see Macmicking etc., Cell.81:641-650 (1995).

To in following non-limiting examples, be illustrated now the present invention.

Embodiment

The present invention will be described referring now to the following example.Provide these embodiment only for illustration purposes, should not think that the present invention is subjected to the restriction of these embodiment and should think that any and all changes that the instruction that provides owing to this paper becomes apparent have been provided in the present invention.

In addition, the inventor has determined that the gene conveying provides the effective means that realizes the lasting and continuous delivering therapeutic agents of eye.The systematicness that embodiments of the present invention will obtain opportune moment of enough gene transfer, enough gene expression, expression and expressed protein for treatment agent and distribution, negligible expressed human cytokines distributes, the suitable biologically active of expressed protein for treatment agent and/or the immune response that does not exist or weaken.Described embodiment will summarize using to carry based on the carrier of the carrier of BIV, described support source based on BIV is from the ox slow virus, and in some embodiments its protein combination with reduction complement activation (for example, alternative route complement activation) is carried.Purpose is treatment and/or research senile macular degeneration (AMD) and two class AMD in latter stage (pattern atrophy and moist AMD).These diseases are that main cause and influence blind in the developed country surpasses 25% over-65s crowd.Based on the inventor's conclusion, i.e. the alternative route of complement activation is the important and general immanent cause of the AMD of form of ownership, and the inventor selects this approach as the treatment target.Some polymorphism in the gene of this conclusion and coding complement Profilin (complement factor H) and statistic correlation consistent (Klein etc., 2005 of increase of the risk of the AMD of development form of ownership; Haines etc., 2005; Edwards etc., 2005; Hageman etc., 2005; Li etc., 2006; Maller etc., 2006; Magnusson etc., 2006; Sepp etc., 2006; And Postel etc., 2006).

Embodiment 1 has summarized some and has generated the method for biv vector by the process that can be used for most of slow virus carriers (if not the words of all slow virus carriers).Embodiment 2～7 illustrated biv vector in vivo to the animal retina cell carry out genetic modification and external to former generation the human retina cell carry out the efficient of genetic modification.These embodiment have also comprised two effect research in the mouse model relevant with the human illness in eye of treatment.Thereby embodiment 8 has described by hindering the novel therapeutic albumen that positive feedback loop reduction complement pathway activates.These albumen comprise three kinds of dominant variants of the complement factor B of being appointed as fB1, fB2 and fB3.Embodiment 9 has described the production of antibodies that suppresses complement pathway.Embodiment 10 and 11 has discussed the external assessment that suppresses the albumen of complement.Embodiment 12 has summarized the strategy of assessing carrier in the mouse model relevant with the AMD of complement-mediated in the body.Embodiment 13 has adopted not carrier system based on slow virus to carry albumen.Embodiment 14 is comprised illustrating that the present invention has function to other disease or illness except that eye disease.Embodiment 15～18 provides additional detail and data partly to suppress people and other inhuman complement pathway with explanation dominant factor B.Embodiment 19 provides factor D cutting dominant factor B partial data.Embodiment 20 has detected the affinity of dominant factor B variant and complement factor C3b formation compound.Embodiment 21 has shown that dominant variant fB3 and Complement Factor D form stable compound.Embodiment 22～24 has illustrated that the antibody capable of anticomplement factor B and D suppresses complement pathway.Embodiment 25 and 26 provides the bioactive data and having described of the people fB3 albumen of checking purifying to be used to generate the generation of the cell-line of people fB3.Embodiment 27 and 28 has described in the complement activation model of mouse damage from laser the assessment to the biv vector of coding people fB3, and in the same animals model to the assessment of people fB3 protein injection.Embodiment 29 provides the detailed and scalable scheme that is used for concentrated and purifying biv vector.Embodiment 30～32 provides the modification to the carrier production decision among the embodiment 29.

The generation of embodiment 1.BIV carrier

Describe some commonsense methods that generate biv vector in the document, for example, seen Matukonis etc., 2002; Molina etc., 2004 and No. the 6th, 864,085, United States Patent (USP) and PCT WO is disclosed No. 03/066810.In some method, can generate carrier with four kinds of compositions.As shown in Figure 2, these compositions comprise: 1) BIV transfer vector construct; 2) expression construct of coding BIV gag/pol polyprotein; 3) coding is as the expression construct of envelope proteins such as VSV-G albumen or baculoviral gp64 envelope protein; With 4) expression construct of coding BIV rev albumen.Described transfer vector construct contains heterologous (therapeutic) gene and produces the rna transcription thing, and described rna transcription thing is wrapped in the carrier granular.Described gag/pol construct and coating construct generate the capsid protein that can form described carrier granular.Described rev construct generates described vector rna is transported the necessary albumen of cell nucleus.

In order to produce carrier, the cell in the medium and described four expression construct are carried out cotransfection by coprecipitation of calcium phosphate.After 1 day～3 days, after described particle is assembled in cytoplasm and has been packed described vector rna, described carrier granular permeate through cell membranes is sprouted, is obtained the lipid bilayer dressing and gathers in tissue culture medium (TCM), can be with its purifying and/or concentrated from described tissue culture medium (TCM).

Particularly, with 1 * 10⁷In individual 293T (ATCC) or 293 FT (Invitrogen) the cell inoculation 150mm ware in the DMEM that adds 10%FBS and at 37℃ 5%CO₂Incubation in the incubation case.Transfectional cell when merge when described plate about 85%～90% next day.Usually, each ware is used the transfer vector construct of 45 μ g, the gag/pol construct of 45 μ g, the coating construct (the baculoviral gp64 envelope protein of encoding in the present embodiment) of 15 μ g and the rev expression construct of 30 μ g.The method that is used for calcium phosphate transfection is known in those skilled in the art, and the calcium phosphate transfection kit is commercially available (for example, Promega).After 24 hours, replace with the medium sucking-off and with the fresh culture that has added the 5mM sodium butyrate.Collect the viral vectors supernatant after 24 hours, filter by 0.45 μ m filter, and with aliquot-80 ℃ of stored frozen to be used for external application.Carrier titre in the cell conditioned medium liquid is generally 2 * 10⁶～6 * 10⁶Individual transduced unit/ml (tu/ml).

For using in the body, can come easily the purified virus carrier and it is concentrated 100 times with supercentrifugation known in those skilled in the art or with following chromatography.The Benzonase of carrier supernatant and 50 units/ml was filtered by 0.2 μ m aPES filter at 37 ℃ of incubations in 30 minutes then.With 300ml carrier supernatant with sample-loading buffer (contain altogether 1M NaCl 2 * PBS) use peristaltic pump with 5ml/ minute speed application of sample on the Sartobind Q75 film adsorber unit (Sartorius) then with dilution in 1: 1.Use then 50ml～75ml lavation buffer solution (contain altogether 500mM NaCl 1 * PBS) to wash described Q75 film adsorber unit in 5ml/ minute.Behind washing step, with the following wash-out of carrier.Described Q75 unit is disconnected and (1 * PBS) the 5ml syringe that contains the NaCl of 1.3M altogether carefully is connected with containing elution buffer from peristaltic pump.Under about 5 droplets/minute speed, collect 3 1ml flow points, the interval is 15 minutes between each flow point of collection.The carrier that concentrates is eluted in flow point 2 and the flow point 3.Via with Vivaspin20 (1 * 10⁶Dalton mwco) (Sartorius) centrifugation apparatus carries out diafiltration and has realized concentrating by the further twice of the buffer-exchanged in store buffer liquid.To concentrate carrier and place in the Vivaspin20 unit, insert the dialysis cup, and add the suitable store buffer liquid of 12ml.This unit is decreased to 0.75ml～1.0ml centrifugal about 40 minutes of 800g or up to the volume that concentrates carrier.To concentrate carrier carries out filtration sterilization with 0.2 μ m PES injection filter and carries out five equilibrium then and be stored in-80 ℃.If described store buffer liquid contain albumen (for example, BSA), then later but add BSA before the aseptic filtration in diafiltration.This step causes the carrier titre to increase about 100 times and output being generally about 30% usually.Unless indicate in addition, described store buffer liquid is to have replenished 2.5mM KCl and 1.0mM MgCl₂PBS.

In the following example, when with slow virus carrier transduction cells in culture, in tissue culture medium (TCM), add the cohesion amine (Polybrene) of 8 μ g/ml or the nucleoprotamine of 1 μ g/ml or 5 μ g/ml.All these polycation additives can exchange use usually.Unless indicate in addition, they are not used jointly with described slow virus carrier when being injected directly into polycation in the animal.

Transduction inembodiment 2. rodent retina cells' the body

Biv vector transduceed in vivo effectively rat and mouse the eye cell.For rat and the mice study shown in Fig. 3 and Fig. 4 A, the GFP carrier is concentrated and it is formulated among the PBS that has replenished 2%BSA by ultracentrifugation.In described carrier, add 8 μ g/ml cohesion amine during injection.For the mice study shown in Fig. 4 B and the 4C, the chromatography by embodiment 1 concentrates described carrier and be formulated among the PBS.Cohesion amine is not used jointly with described carrier.Be about 1 * 10 in subretinal injection 1 μ l～3 μ l titres⁸The carrier of the encoding green fluorescent protein (GFP) of tu (transduced unit)/ml detects GFP to described retina subsequently and expresses.Described rat was tracked to many 9 months, and described mouse was tracked to many 5 months.In two animal models, seen that in the retina cell at injection site place high-caliber GFP expresses.In addition, during each research, there is not perceptible expression loss.The example that GFP in the rat retina expresses as shown in Figure 3, and the example that the GFP in the mouse retina expresses is as shown in Figure 4.Immunocytochemical assay to the retina cross section shows the mainly expression in retinal pigment epithelium (RPE) cell (retina cell's type of photosensitive layer below) of described carrier.

Transduction inembodiment 3. rabbit retina cells' the body

Chromatography by embodiment 1 has prepared the biv vector of coding GFP and it has been formulated into the PBS/KCl/MgCl of the embodiment 1 that is supplemented with 50mM trehalose and 0.1%BSA₂In the buffer solution.With the anesthesia of the New Zealand white rabbit of every heavily about 3kg, Tropicamide with 1% and 1% AK expander (dilate) make pupil dilation.One 0.5% proparacaine instils in every eye.Place eye speculum, and with povidone iodine wiping eyelid and conjunctival cul-de-sac.Under the Zeiss surgical operation microscope, carry out centesis to reduce intraocular pressure with the 30g syringe needle.Haptic lens is placed on the cornea to help to observe retina.With 25g syringe needle (SurModics) in STQ with steep angle push through conjunctivae and selerae to the corneal limbus 3mm～4mm to avoid eyeglass.The 39g sleeve pipe is guided by described 25g syringe needle up to slightly touching retinal surface.Pulse injection 100 μ l titres are 1 * 10⁸The GFP carrier solution of tu/ml cuts to realize retina.Clearly formed blister under the retina.Fall pin then and sleeve pipe slowly takes out from eye, this has caused the scleral incision of automatic sealing.After described operation, animals received the subconjunctival injection of Kenalog-G of 0.5ml, and the cornea that contains the ointment of neomycin, polymyxin B and dexamethasone (Alcon) is used.

All around, put to death described rabbit and collect retina, prepare the full sample of retina then.As shown in Figure 5, in the amphiblestroid RPE cell of rabbit, the stronger expression of GFP is arranged.Express in the neural retina that also sees various cell types (data not shown).

Transduction inembodiment 4. monkey retina cells' the body

Chromatography byembodiment 1 has prepared the biv vector of coding GFP and it has been formulated into (20mM HEPES pH 7.4,130mM NaCl, 1mM MgCl in the HEPES buffer saline₂, the 50mM trehalose, 0.1%BSA).The carrier titre is 1.5 * 10⁸Tu/ml.With making the BIV GFP carrier of having accepted 75 μ l in each comfortable eye of two machins by subretinal injection to the described similar operation method of rabbit (embodiment 3).After 10 weeks monkey put to death and prepare smooth retina sample.Fig. 6 has shown that the GFP in the amphiblestroid RPE layer expresses.

GFP in theembodiment 5. former generation people RPE cells expresses

Obtain human eye from the Lions eye bank of Oregon from 75 years old women, 73 years old male sex and 43 years old male sex.By removing leading portion, vitreum and neurosensory retina described eye is dissected.Eyecup cleaned with PBS and in 0.05% trypsase-EDTA of 2ml according to the situation incubation ofRPE layer 10 minutes～20 minutes.With scraper the RPE cell is gently scraped and is collected in the 15ml conical tube.Described cell is washed centrifugal 5 minutes of 100rpm and with PBS.At last, will be resuspended among the DMEM+15%FBS of 1ml, be inoculated into then in 12 well culture plates from the RPE cell of each.

The RPE cell is slowly grown and is spent and 2 week～3 weeks became fusion in the hole.In order to determine that chromatophore is RPE, they have been tested the expression of RPE specific proteins RPE65 by immunofluorescence dyeing.As shown in Figure 7, described cell shows strong dyeing to the monoclone antibody (Novus Biologicals) of RPE65.On the contrary, in omitting a contrast that resists, only observe background dyeing (data not shown).

In the presence of 2 μ g/ml nucleoprotamine with the BIV GFP of the 5 μ l described cell of transduceing.Described carrier is formulated among the PBS and has 1x10⁸The titre of tu/ml.As shown in Figure 7, the GFP that back 48 hours described cell displays go out mark that transduces expresses.

Embodiment 6. can induce the VEGF model

Shown in above-mentioned embodiment, the biv vector retina cell that can in small animal model that comprises non-human primates and large animal model, transduce in the body.And biv vector can be at external effective transduction human retina cell of former generation.Studies show that additionally that described carrier can be transduceed and comprise fibroblastic other cell type of keratocyte, conjunctival cells and sclera (data not shown) in mouse and rat, in described extra research by intravitreal injection, capsula bulbi under injection and periocular injections and use described carrier by on cornea, directly applying described carrier.These data have been supported the gene conveyance strategy to the eye cell, for example, so that the eye disorders of improvement or stabilizes.

Carried out several researchs so as the checking biv vector can with treatment people's eye disease diseases associated model in realize usefulness.In following two embodiment, thus described vector encoded anti-angiogenesis gene blocking-up or suppress neovascularity growth and seepage in retinal vessel new life's the mouse model.After being expelled to the mouse eye, described carrier carries out genetic modification to the retina cell.Emiocytosis anti-angiogenesis after modifying then, described anti-angiogenesis diffusion spread all over whole eye and block and/or suppressed angiogenesis.

In the ocular angiogenesis newborn mice model of high far, assessed the biv vector (for example, seeing Okamoto etc., 1997) of coding anti-angiogenic proteins Endostatin (Endostatin cDNA available from InvivoGen, catalog number (Cat.No.) pbla-hendo18).Thereby described mouse through engineering approaches its photoreceptor when it is handled with vibramycin is begun secretion of VEGF.In 3 days, VEGF causes significant vascular leakage and neovascularization.In one week, the serious consequently retina of pathology comes off from the eyeground.

In every animal, use the control vector treatment another eye (Takahashi etc., The FASEBJournal, online publication on March 28 in 2003) of the treatment albumen of not encoding by eye of subretinal injection treatment with BIV Endostatin carrier.After 3 weeks, use vibramycin.After 5 days, the vascular leakage in the live animal is assessed by FA.

In this assessment, fluorescein is used by intravenous injection, and made the vascular leakage in the retina visual by the diffusion phosphor pattern in the eyeground.

After handling seven days, vibramycin puts todeath 10 mouse of independent cohort.The retinal tissue cut sections for microscopic examination by thickening that vascular leakage causes, and are checked the serious consequence of retinal detachment to whole eye cross section.

The result is depicted among Fig. 8 and is provided in (2003) such as Takahashi.FA has disclosed widely vascular leakage but has disclosed normal in the eye of Endostatin vehicle treatment or near normal vascular patterns in contrast eye.Histological assessment has confirmed these results.Show seriously thickening that vascular leakage causes from the retina of contrast eye, yet seem to thicken normal or minimumly from the retina of the eye of Endostatin vehicle treatment.At last, the contrast eye suffers retinal detachment partially or completely, yet the eye of Endostatin vehicle treatment demonstrates the retinal detachment of much less and do not have retinal detachment in some cases.In general, BIV Endostatin carrier has been protected subject eye in 80% the mouse.And described result of treatment expansion spreads all over each retina and is not limited to injection site.

These data show that biv vector has the potentiality of prevention ocular angiogenesis new life and seepage.

Embodiment 7. damage from laser models

In ocular angiogenesis new life's laser loss model, assessed and had two kinds of genetically modified biv vectors of difference.Start from and be accepted this model that is used to develop a therapeutic agent and (for example, see Gehlbach etc., Hum Gene Ther.200314 (2): 129-41 to stimulate angiogenesis with laser burn retina; Mori etc., Invest Ophthalmol Vis Sci.200243 (6): 1994-2000; With Mori etc., J CellPhysiol.2001 188 (2): 253-63).In brief, thus described burn in retina pore-creating make new capillary can from below the CC bed grow in the retina.Common defectiveness of described blood vessel and seepage.

In first research, be administered to the rat intraocular by will the encode biv vector of anti-angiogenic proteins pigment epidermal derived factors (PEDF) (cDNA of PEDF available from InvivoGen, catalog number (Cat.No.) pbla-hpedf) of subretinal injection.In every rat, eye is accepted described PEDF carrier and the another eye accepts not encode the genetically modified contrast biv vector of therapeutic.After two weeks, by damage from laser induction of vascular new life.After two weeks, handle described rat with the FITC-glucan that can describe the neovascularity profile.Collect retina and inspect blood vessel structure.The results are shown among Fig. 9.As expection, the eye for the treatment of with control vector shows pathological.Otherwise, in the outward appearance that has with the new capillary in the eye of described PEDF vehicle treatment as the feature of decomposing angiogenesis.

In second research, in the damage from laser model mice, assessed the biv vector of coding anti-angiogenic proteins T2-TrpRS (cDNA of T2-TrpRS available from InvivoGen, catalog number (Cat.No.) pbla-htrprs).The eye of every mouse is accepted described T2-TrpRS carrier, and the another eye is accepted control vector.On the same day, by laser loss induction of vascular new life.After two weeks, assessed the size in new vessels zone with FITC-glucan and serial section.Thereby the data show among Figure 10 is very effective in angiogenesis inhibiting with obviously littler this carrier that shows in mean nascent blood vessel zone in the eye of T2-TrpRS vehicle treatment.

In addition, supported to be used to improve from the data of these PEDF and T2-TrpRS zooscopy and/or the application of the gene transfer of stable eye disease.

Can the weaken design of treatment albumen of complement activation ofembodiment 8.

Obtained to have the wild type people fBcDNA of its natural flanking sequence at 5 ' and 3 ' end from human liver cDNA library (Origene Technologies, Inc., catalog number (Cat.No.) CH1005) by PCR.Two primers that are used for PCR are: 5 '-CTAGCTAGCTCCTGCCCCAGGCCCAGCTTCTCTCC-3 ' (forward primer) (SEQ ID NO:17) and 5 '-CTAGCTAGCTCAATCCCACGCCCCTGTCC-3 ' (reverse primer) (SEQ ID NO:18).Two primers all contain Nhe I site.With the PCR product after the amplification with Nhe I digestion and be connected in advance in the BIV metastasis transplanting physique grain that digests with Nhe I (seeing embodiment 15).In this construct, fB transcribes with the MNC promoters driven.The sequence of described carrier and fB obtains confirming.The dna sequence dna of wild type people fB is shown in SEQ ID NO:1, and amino acid sequence corresponding is shown in SEQ ID NO:2.Shown illustrative among Fig. 2 to the lentivirus transfer vector construction body of coding fB.In the case, the construct that is designated as " heterologous gene " partly is described fB sequence.The described nucleotide sequence that should be noted that sequence list comprises flanking sequence.

Subsequently, the dna sequence dna of directly synthetic 3 people's dominant fB mutant/analogs (fB1, fB2 and fB3) of encoding.Except the specific sudden change in the fB code area, these three dna sequence dnas are identical with SEQ ID NO:1.At amino acid levels, fB1 contains D740N to be changed.The dna sequence dna of fB1 and amino acid sequence are respectively shown in SEQ ID NO:3 and SEQ ID NO:4.At amino acid levels, fB2 contains D279G, N285D and D740N changes.The dna sequence dna of fB2 and amino acid sequence are respectively shown in SEQ ID NO:5 and SEQ ID NO:6.At amino acid levels, fB3 contains K258A, R259A, K260A, D279G and N285D to be changed.The dna sequence dna of fB3 and amino acid sequence are respectively shown in SEQ ID NO:7 and SEQ ID NO:8.With each dominant people fB construct as Nhe I fragment subclone in the BIV metastasis transplanting physique grain.All constructs are checked order to verify its integrality.

Directly synthesized inaddition 3 similar mouse dominant fB mutant/analogs (Geneart, Inc.) and with they as Nhe I fragment subclone in the BIV metastasis transplanting physique grain.Mouse fB1 contains D737N to be changed.Its sequence is respectively shown in SEQ ID NO:9 and SEQ ID NO:10.Mouse fB2 contains D276G, N282D and D737N changes.Its sequence is respectively shown in SEQ ID NO:11 and SEQ ID NO:12.Mouse fB3 contains K255A, R256A, K257A, D276G and N282D to be changed.Its sequence is respectively shown in SEQ ID NO:13 and SEQ ID NO:14.Obtain mouse wild type fB by reverse engineered to mfB1.Particularly, by rite-directed mutagenesis 737 N is converted into D with Quick Change kit (Stratagene Inc.).The sequence of mouse wild type fB is respectively shown in SEQ ID NO:15 and SEQ ID NO:16.All constructs are checked order to verify its integrality.

Herein, people's factor B wild type and mutant/analog are appointed as are had alphabetical h (for example hfB1) and the mouse analog is formulated to having alphabetical m (for example mfB1).

Such asembodiment 1 general introduction generated the slow virus carrier goods, and described carrier is expressed assessment external.With carrier supernatant transduction ARPE cell (human retina pigment epithelial cell system (ATCC, CRL-2302)).Transduction with the biv vector pair cell is being described (for example, Matukonis etc., 2002 before this; Molina etc., 2004).In brief, with the ARPE cell with everyhole 1 * 10⁵The density cover plant of individual cell is in 6 orifice plates.Next day, in the presence of 5 μ g/ml protamine sulfates (Sigma) at 37℃ 5%CO₂In the incubation case with the 3ml carrier supernatant described cell of transduceing.After 5 hours, (DMEM ofband 10%FBS) replaces medium with fresh cell culture medium.With the cell culture 72 hours after the transduction, at this moment medium is carried out SDS-PAGE and the Western trace is expressed to analyze fB.

40 μ l cell culture mediums are mixed and be heated to 95 ℃ with 5 * SDS-sample buffer of 10 μ l reach 3 minutes.Then sample is separated on the 7.5%SDS-polyacrylamide gel.Albumen after separating is transferred on the nitrocellulose filter, described film is detected with the anti-people fB of goat serum (NordicImmunological Laboratories, catalog number (Cat.No.) GAHu/PFB).Then with described film and the anti-goat IgG of biotinylated rabbit (Vector Laboratories, catalog number (Cat.No.) BA-5000) incubation then with avidin-biotinylated alkaline phosphatase multienzyme complex incubation.At last, described film and alkaline phosphatase substrate (Vector Laboratories) incubation so that band are manifested.People's wild type fB, fB1, fB2 and fB3 representative data are shown among Figure 13.The correct fB albumen of molecular weight is secreted with the level of 2.5 μ g/ml from the cell of the transduction of suppressed by vector.Carrier with encoding murine wild type and dominant fB albumen has obtained similar data (data not shown).These data show biv lentiviral vector can mediate fB albumen in the retina cell effective expression and support potential in clinical application.

Embodiment 9. suppresses the binding molecule strategy of complement fB activity

The second kind of strategy that suppress to substitute complement pathway relates to the antibody (for example, monoclone antibody) that can neutralize or suppress the fB activity or the generation of its fragment.Monoclone antibody is conventional the generation in mouse, rabbit and chicken.For the present embodiment, the inventor has used rabbit.The possibility of acquisition with the monoclone antibody of the mouse fB cross reaction of rodent research usefulness optimized in the use of rabbit.Obviously this has simplified the research in rodent, but this is not the necessary condition that suppresses people fB.Exemplary policy and then relate to from hybridoma and for example come the clonal antibody sequence so that produce the single-chain antibody of reorganization via RT-PCR.The technology of preparation single-chain antibody is (Carolina etc., 1994) known in those skilled in the art.In case of necessity, equally by well known to a person skilled in the art that method (Adams etc. 2005) carries out humanization to the framework sequence, thereby can further optimize described antibody to be used for the treatment of purposes.In addition, can change antibody or antibody fragment in conjunction with character, for example, see United States Patent (USP) the 7th, 175, No. 996 and the 6th, 656, No. 467.At last, antibody (for example, and fB single-chain antibody) can be coded in the carrier and by gene transfer and carry.In some embodiments, can carry antibody or its fragment.

The process of rabbit monoclonal antibodies that produces anti-people fB is as follows.From Quidel (San Diego, CA) the people fB in (＞95%) plasmid source of acquisition purifying.People fB with total amount 2.5mg reaches 3 times every rabbit immunity.After immunization protocol begins 6 week～3 month, the blood serum sample from described rabbit is detected the antibody titer of anti-fB through ELISA.Also to described blood serum sample in haemolysis test (seeing embodiment 11) in and titre test.Thereby the test of human specific haemolysis and mouse specificity haemolysis test announcement antibody and people fB and mouse fB cross reaction have been carried out.The rabbit that has high titre serum is used to produce monoclone antibody.Remove spleen to be used for cytomixis.Separate hybridoma, will from the monoclone antibody of each hybridoma through ELISA at people fB and mouse fB in and titre screen.1 time～3 times the stability of hybridoma subclone that secretion had the monoclone antibody of senior middle school and titre to guarantee that antibody generates.Along with each subclone, in and titre screening and cloning body.Choose the RT-PCR amplification that best clone body is used for producing the antibody sequence of recombinant single chain antibody then.In brief, primer is designed to the flank at the antibody variable region of giving antigen-binding specificity.Then pcr amplification is carried out in described variable region and the gained dna sequence dna is used to make up the single-chain antibody that has general structure shown in Figure 14.The recombinant single chain antibody of reappraising still demonstrates fB combination and neutralization activity to guarantee them.Then with described single-chain antibody subclone in the transfer vector construct of Fig. 2 in case produce biv vector be used for external and body in assess.Can further modify the single-chain antibody of the clinical practice that is selected to the people so that the framework region humanization.At last, adopting the orthogenesis technology in case of necessity also further improves among the fB and usefulness (for example, seeing Broder etc., 2000) so that further increase described antibody to the affinity of fB.These technology include but not limited to that CDR transplants, framework is reorganized and resurfacing (resurfacing) technology.

In some embodiments, with indivedual CDR from described hybridoma pcr amplification and be attached in the DNA construct of polypeptide of coding band antigen binding capacity.Though these polypeptide do not have the structure of single-chain antibody, they effectively in and fB and serving as need the albumen (for example, treatment albumen) carrying or carry as albumen through biv vector.

Produce although the antibody in the present embodiment is the people fB at plasmid source, also can even produce the antibody of effects equivalent by the fB polypeptide of making (for example, 20 amino acid epi-positions) by reorganization fB.At last, can utilize same or analogous tactful generation can in and the antibody of any composition of complement pathway.Complement Factor D (fD) is to substitute the excellent target that the antibody strategy of complement pathway is used in order to weaken, and this is because fD appears in the blood plasma with extremely low-level (about 1 μ g/ml～2 μ g/ml) and in the eye and mediated the rate-limiting step (Volanakis in the alternative route; Narayana etc., 1996 ProteinScience 5:553-564).And, in and the recombinant single chain antibody of fD can use with the technical combinations of above-mentioned reduction fB activity.

The step that rabbit immunity, hybridoma generation, subclone and antibody are collected can be undertaken by commercial entity (as the Genesis Biotech in Taiwan, Inc. etc.).In addition, many companies provide the service (for example, Covance or Charles River Laboratories) of producing mouse monoclonal antibody.

The haemolysis test of the example ofembodiment 10. assessment fB dominant bodies

Use the biv vector of coding people wild type fB and the described three kinds of dominant bodies ARPE cell of transduceing, and with the functional activity of the secreted fB albumen of the haemolysis testing evaluation of alternative complement pathway.This test utilization is spontaneously activated the rabbit erythrocyte (Sohn etc., 2000) of the alternative route of people and mouse.At first, in the interpolation of pH value 7.35 Mg⁺⁺With prepared 2 * 10 in gelatin/veronal (veronal) buffer saline (GVB-EGTA) of EGTA⁸The cell suspension thing of the not sensitization rabbit erythrocyte (Erab) of individual cell/ml.Secondly, 20 μ l have been exhausted the human serum (Quidel) of fB dilutes by 1: 5 with GVB-EGTA, described GVB-EGTA contains: (1) is additive-free; (2) the people fB (Quidel) in the blood plasma of 500ng source; (3) control oneself by 40 μ l medium of the ARPE cell of the carrier transduction of coding GFP, wild type people fB, fB1, fB2 or fB3.Described medium has been used Amicon ultracentrifugation filter (Millipore, catalog number (Cat.No.) UFC803008) to carry out 10 times and has been concentrated.Then each serum mixture of 100 μ l is added among the Erab of 100 μ l and incubation 60 minutes in 37 ℃ oscillator water-bath.With ice-cold NaCl (0.15M) stopped reaction.Withpipe 4 ℃ centrifugal 10 minutes of 1250g so that cell precipitation, and determine the OD of each supernatant₄₀₅For over against photograph, in the Erab suspension, add distilled water, this causes 100% infiltration cracking of described cell.

Data are presented among Figure 15.In the time of in joining the serum of exhausting fB, the people fB in 500ng purifying blood plasma source has produced 80% haemolysis (100% comparing with cracking obtains by infiltration).The serum of exhausting fB that does not add any fB does not produce haemolysis.The tissue culture medium (TCM) of cell of GFP carrier transduction of using by oneself does not recover the haematolysis ability of serum.Otherwise the tissue culture medium (TCM) of the cell of the wild type people fB carrier transduction of using by oneself has produced 40% haemolysis.Be that the tissue culture medium (TCM) of the cell of the carrier transduction of each does not recover the haematolysis ability of serum in the described three-type-person's dominant fB part of the coding of using by oneself as expected.These data verifications people's wild type fB in carrier source be bioactive and the dominant body does not activate alternative complement pathway.

Embodiment 11. utilizes the haemolysis testing evaluation to suppress the albumen of complement pathway

The competition test can be used to assess tiring of coding dominant fB carrier partly.In tissue culture, prepare fB dominant albumen by carrier transduction to the ARPE cell.FB albumen is secreted in the tissue culture medium (TCM) and by the analysis of the Western shown inembodiment 8 it is carried out quantitatively.The test that is at war with has wherein added the dominant fB and the wild type fB of various ratios in the haemolysis test.Dominant fB can rebuild the ability of the hemolytic activity of the serum of having exhausted fB with wild type fB protein competition and the wild type fB albumen that weakens, and has determined tiring of they according to the ability of every kind of dominant fB.Also can in animal model, assess the carrier of encoding said proteins, and can further develop in some cases and have expectation (for example, the most effective) carrier of tiring, for example be used to study complement pathway and/or the application in animal (for example, people) etc.

Indicate as this paper, it is believed that fB1 can be with normal affinity and dynamics in conjunction with C3b, but fB1 can not bring into play protease function and not form C3 convertase when being subjected to the fD effect and being stablized by properdin.FB2 have increase to the binding affinity of C3b and make the proteinase activity inactivation simultaneously.FB3 have increase to the binding affinity of C3b but can not be cut by factor D, therefore should have minimum proteinase activity.In testing, fB1, fB2 and fB3 are tested with the competition of wild type fB.

To the described similar haemolysis test ofembodiment 10 in assessed tiring of anti-fB antibody with the serum of having exhausted fB.Particularly, described measurements determination the dilution of every kind of antibody/concentrate blocking-up or suppress purifying fB and rebuild tiring of hemolytic activity ability.The carrier that does not adopt the described antibody of generation to use with antibody protein has carried out the initial external assessment of described anti-fB antibody in described haemolysis test.That is, in described test, add antibody purification and weaken tiring of haemocylolysis to assess various antibody.Use subsequently from the tissue culture medium (TCM) of ARPE cell by the external assessment of haemolysis test carrying out, described ARPE cell is by the carrier transduction of the antibody (for example, the most effective antibody) of coding single stranded form.

The body inner analysis of the carrier ofembodiment 12. coding fB dominant bodies and fB neutralizing antibody

At first with mouse fB dominant body and in and those antibody of mouse fB in mouse, carry out assessing in the body.Also in mouse, assessed people fB dominant body in the body, but species specificity may disturb the reliability of these assessments.

The mouse model that is used to assess the carrier of the described potential treatment albumen of coding is damage from laser model (Campochiaro and Hackett 2003).As described in embodiment 7, with laser pulse pore-creating in Bruch's membrane, the new blood vessel of growing from the CC bed passes described hole.Carried out quantitatively the degree of angiogenic growth by FITC-glucan infusion after week laser treatment 1 week～2.What is interesting is that the growth of neovascularity depends on the activation that substitutes complement pathway.When nearest data show was suppressed in mouse or blocks when alternative route, the angiogenesis of induced with laser significantly reduced (Bora, N. etc., 2006; Bora, P. etc., 2006; And Bora, N. etc., 2007).Therefore, this model provides and has been used to assess the short-cut method that gene transfer vector of the present invention suppresses the validity of complement activation in vivo.Since described model measurement the complement inhibition, so it has predictive value for the exploitation of therapeutic agent that the treatment teiology relates to the human diseases of complement activation, described disease comprises the AMD (early stage dryness AMD, moist AMD and ground pattern atrophy) of form of ownership.At last, thus it should be noted that complement component C3 a and C5a in the drusen that is found in the people according to the show can the outer vegf expression of inductor shows that mouse medium vessels new life's mechanism may similar to the mankind (Nozakideng etc., 2006).

In brief, with the control vector of the carrier of coding fB dominant body or the irrelevant albumen of coding (for example, do not encode and treat the carrier of albumen) be expelled to (for example, new life's (p5) or puberty (about 6 ages in week) C57B1/6 mouse) in the mouse by subretinal injection or intravitreal injection.When about 8 weeks of described animal during ages, produce 3 points at each retina thereby carry out damage from laser.Many different damage from laser methods are well known by persons skilled in the art.The inventor uses Iris OculightSLX laser instrument usually, and this laser instrument contains red diode and emission wavelength is the light of 810nm.Usually laser parameter is set for beam diameter and is 75 μ m, energy level and be 100 milliseconds of 100 milliwatts and pulse durations.After the damage fromlaser 7 days, pour into described animal, collect retina and determine the angiogenesis degree by Laser Scanning Confocal Microscope with the FITC-glucan.

For the test antibody strategy, carry out damage from laser in the C57B1/6 mouse in puberty.On the same day by the described animal of the mab treatment of the anti-fB of intravitreal injection.In every kind of situation, to the irrelevant Antybody therapy of negative contrast cohort.

For analyzed in vitro, usually but may not always be to use the hybridoma of expressing potent antibodies to produce recombinant single chain antibody.Then these single-chain antibodies are encoded in the gene transfer vector, and with the described identical mode of fB dominant body is tested in mouse.Use for the mankind, make the framework region humanization thereby the anti-fB single-chain antibody of reorganization further can be modified where necessary.

Adopt the orthogenesis technology in case of necessity so that further increase described antibody to the affinity of fB with further improve among the fB and usefulness (Broder etc., 2000).

The non-carrier system based on slow virus of embodiment 13. usefulness is carried fB dominant body and anti-fB antibody

Although previous embodiment concentrates on lentiviral vector genome transfering system (being specially the carrier system based on BIV), yet also can albumen or therapeutic agent be transported to eye by other carrier system.For example, above-mentioned dominant fB part and single-chain antibody are easy to be encoded in the AAV carrier.The use of AAV carrier is very easy to and is to well known to a person skilled in the art (for example, to see Lu 2004; United States Patent (USP) the 7th, 037, No. 713, the 6th, 953, No. 575, the 6th, 897, No. 063, the 6th, 764, No. 845, the 6th, 759, No. 050, the 6th, 710, No. 036, the 6th, 610, No. 290, the 6th, 593, No. 123, the 6th, 582, No. 692, the 6th, 531, No. 456, the 6th, 416, No. 992, the 6th, 207, No. 457 and the 6th, 156, No. 303).Have at least 8 kinds to have different vivo gene transfer efficient and the initial AAV serotype of different expression.Most of AAV carrier serotypes can in eye, play a role (for example, seeing Aurricchio etc., 2001 and Yang etc., 2002).(La Jolla CA) is purchased the AAV carrier system that attaches detail specifications can to pass through Stratagene.With treatment albumen subclone to described carrier in, be easy to and be well known to a person skilled in the art by transient transfection generation carrier products and the step by density gradient ultracentrifugation or the described carrier of column chromatography purifying.The external AAV carrier of having assessed encoding proteins (for example, albumen described in the previous embodiment) and with its by with the described same or analogous method of biv vector is expelled in the animal model.

Embodiment 14. the present invention are treating as the application in the human diseasess such as atherosclerotic cardiovascular disease

Although previous embodiment concentrates on eye disease, it should be noted that many different human diseasess are the cause of disease with the complement activation.These diseases include but not limited to that rheumatism, neuropathy and cardiovascular disease or the like (for example, see Niculescu and Rus 2004; Kardys etc., 2006; With Rus etc., 2006).Therefore, some carrier of the present invention has inhibition, stablizes and/or treat the instant application of other disease except that illness in eye, for example by carrier of the present invention or albumen are injected directly in the ill organ.Particularly, atherosclerotic plaque can be at least in part by with to the described identical teiology growth of the drusen in the AMD model provided herein.The local expression of complement inhibitor and/or conveying will slow down the development of patch and slow down the progress of disease.In some embodiment of treatment coronary artery disease or peripheral arterial disease, can be with vector administration to blood vessel.If treatment relates to angioplasty, then can carrier and/or albumen be administered to for example angiopoiesis site by conduit.If treatment relates to vasotransplantation, then can before transplanting, carrier and/or albumen be passed through vascular infusion.In some embodiments, carrier and/or the albumen recurrence or the progress that can weaken local inflammation and slow down atherosclerotic plaque.Figure 16 provides support to non-ophthalmic applications with gene transfer to the efficient of blood vessel and brain by showing slow virus carrier.

Figure 16 shows that the lentiviral vector genome to rat aorta and mouse brain shifts.Figure 16 A has showed the transduction of part rat aorta.In this case, with the slow virus carrier that is derived from HIV of expressing beta galactosidase blood vessel is carried out infusion.With beta galactosidase reporter protein through engineering approaches so that navigate in the cell nucleus.To described Vascular Slice and to beta galactosidase dyeing, described dyeing produces blue immediately.The nuclear of the blueness shown in Figure 16 A has been indicated gene effectively to transfer to along the endothelial cell on surface, chamber and smooth muscle cell and has been seen through vascular wall to a certain extent.The generation of HIV carrier and application are well known to a person skilled in the art, and the HIV carrier system can (Carlsbad CA) be purchased from Invitrogen.Figure 16 B has showed with the gene transfer of BIV GFP carrier to mouse brain.1 μ l carrier is used black substance by stereotactic injection.Put to death mouse after 17 days and with brain section.GFP expresses and is found in neuron and the spongiocyte.What is interesting is, even, can find that still GFP expresses in the brain both sides only at a side injection of brain.With the neuron dyeing of NeuN, with red display to brain.Thereby illustration is presented under the high power yellow coincidence of green dyeing and red staining and has verified neuronic transduction.

In addition, above-mentioned analyzed in vitro and body inner analysis are united the gene transfer strategies of having supported to treat with the gene transfer vector of coding anti-inflammatory treatment albumen human illness in eye.The research of present embodiment shows that also the present invention treats the potentiality as other human diseasess such as cardiovascular disease and neuropathy.

Embodiment 15. people's factor B mutant substitute the inhibition of complement pathway activity to the people

Material, method and apparatus

Beckman Allegra 6KR centrifuge, C76 water bath chader (New BrunswickScientific Classic Series),Fisher Vortex Genie 2, Molecular Device SpectraMax 190 microplate reader, Corning 96 orifice plates (white), 14ml polystyrene round bottom pipe (Fisher), molecular weight are held back the Amico ultrafiltration apparatus (Millipore) into 30K.

Reagent and buffer solution

GVB⁺⁺(Sigma) contain 5mM barbitol buffer solution, 0.15mM CaCl₂, 141mMNaCl, 0.5mM MgCl₂Mg²⁺-EGTA buffer solution prepared fresh and contain 100mM EGTA, 100mM MgCl when each the use₂, GVB⁺⁺With 5% glucose.Rabbit erythrocyte is available from Innovative Research.The serum of having exhausted people's factor B is available from Quidel (catalog number (Cat.No.) A506).

Generation by people's factor B wild-type protein of biv vector coding and three kinds of dominant people mutant protein fB1, fB2 and fB3

The structure plasmid is used to produce the carrier based on BIV.PAVTrGP038 (SEQ ID NO:19; Figure 29 A) has the RSV promotor, described RSV promotor operably is connected with BIV gag/pol code area (threonine in the DTGAD motif of encoding proteins enzyme is to the sudden change of serine) and is thereafter synthetic poly a-signal, for example, sees United States Patent (USP) the 7th, 070, No. 993.The codon optimized of gag/pol coded sequence expressed being used in addition, for example, see Molina etc., Hum Gene Ther.2004 15 (9): 865-77.PAVTrREV039 (SEQ ID NO:20; Figure 29 B) contain the RSV promotor, described RSV promotor operably is connected with BIV rev code area (by optimum codon recompile) and is thereafter synthetic poly a-signal.PAVTrGP64-040 (SEQ ID NO:21; Figure 29 C) coding GP64 coating.The transfer vector based on BIV of coding people wild type factor B, fB1, fB2 and fB3 (being respectively SEQID NO:2,4,6 and 8) all passes through its separately code area (seeing SEQ ID NO:1,3,5 and 7 code area and embodiment 8 respectively) is cloned into pAVT001 (SEQID NO:22; Figure 29 D) prepares in the Nhe I site.

In order to generate the biv vector particle of encoding wild type people factor B and three kinds of dominant people factor B mutant, with the 293FT cell with 1.1 * 10⁷Individual cell/ware cover plant is in the 150-mm ware.Next day is as cell as described in GP64 env expression construct (pAVTrGP64-040) transfection of the Rev expression construct (pAVTrREV039) of the transfer vector construct based on BIV based on the encoding wild type people factor B albumen, fB1, fB2 or the fB3 that pack construct (pAVTrGP038), 45 μ g of BIV of usefulness 45 μ g as described in theembodiment 1,30 μ g and 15 μ g.Use pAVTGFP006 (SEQ ID NO:23 similarly; Figure 29 E) the contrast biv vector of preparation coding eGFP.After the transfection 36 hours, collect the carrier supernatant and under 4 ℃ centrifugal 10 minutes of 2000rpm to remove all cells chip.

In order to produce wild type people factor B and three kinds of dominant people factor B mutant proteins, contain the tissue culture medium (TCM) transduction 2 * 10 of suitable carrier with 3ml⁵Individual ARPE cell or Cf2Th cell.In contrast, with the carrier transduction cell of 3ml coding eGFP.In order to strengthen transduction, adding ultimate density in the hole is the protamine sulfate of 8 μ g/ml.After 6 hours, replace with the sucking-off of carrier supernatant and with the new fresh cell DMEM medium that 3ml contains 10% heat-inactivated fetal bovine serum.After cell reaches fusion, medium is replaced with the no phenol red medium that 1.5ml contains 2% heat-inactivated fetal bovine serum.After 96 hours, the collecting cell medium also passed through 0.2 μ m filter then with the scavenger-cell chip in centrifugal 10 minutes at 2000rpm and filters.The Millipore Amico ultrafiltration apparatus of holding back to 30K with molecular weight concentrates 5 times with the cell culture medium of collecting then.Assessed the protein expression level of wild type people's factor B and three-type-person's dominant factor B mutant by the Western trace, and found that the expression of described albumen is basic identical, for example, by estimating in 2 times.Store for future use with aliquot with the medium aseptic filtration that contains wild type and dominant negative mutant people factor B albumen after concentrating and at-80 ℃.

Substitute the method for complement pathway hemolytic activity test

Below test is to utilize the competition of the human serum of exhausting factor B test.This has exhausted that the serum of factor B itself does not have detectable complement-mediated hemolytic activity.In described test, add people's wild type factor B and can rebuild the serum hemolytic activity.Contain simultaneously dominant factor B albumen (fB1, fB2 or fB3) culture supernatants interpolation in case show the dominant body whether with the reconstruction of wild type factor B competition and reduction hemolytic activity.

1ml rabbit erythrocyte suspension (Erab) is transferred in the 50ml taper centrifuge tube also with the fresh cold Mg that makes of 30ml²⁺The washing of-EGTA buffer solution.The BeckmanAllegra6KR centrifuge of closing with brake with Erab at cold Mg²⁺In-EGTA the buffer solution centrifugal 5 minutes of 4 ℃, 1200rpm.Erab is washed 2 times and is resuspended in the ice-cold Mg of 2ml again²⁺In-EGTA the buffer solution.Carry out cell counting with hemacytometer.Should be noted that EGTA has brought into play the function that suppresses CCP and do not influenced alternative complement pathway.

First group of research is designed to show that fB1, fB2 and fB3 itself can not rebuild hemolytic activity.Second group of research is designed to show that fB1, fB2 and fB3 have blocked the ability that wild type fB rebuilds hemolytic activity.On ice the hemolytic reaction mixture is placed in the 14ml polystyrene round bottom pipe.For described first group of research, in each pipe, add the medium that contains wild type fB, fB1, fB2 or fB3 that 40 μ l prepare as mentioned above.For described second group of research, in each pipe, add 40 μ l and contain the medium of wild type fB and contain the mixture of the medium of fB1, fB2 or fB3 with indicated ratio.Then, in each pipe, add the human serum of exhausting people's factor B of 25 times of dilutions of 50 μ l.With the described human serum of factor B of having exhausted at the fresh ice-cold Mg that makes²⁺Dilute in-EGTA the buffer solution.With the abundant vortex of described effective Fisher Vortex Genie 2 devices.Then will be through Mg²⁺What-EGTA washed contains 5 * 10⁷Individual erythrocytic 10 μ l Erab add in each pipe, then not vortex and gentle the mixing.The revolution with 110rpm/ minute in 37 ℃ of water-baths of described pipe was shaken incubation 40 minutes.Then, each pipe is placed on ice and add 0.9% ice-cold salt solution of 150 μ l with stopped reaction.Gentle the mixing of described pipe is incorporated in the Beckman centrifuge of closing brake centrifugal 5 minutes of 4 ℃, 2000rpm.Not disturbance precipitates and transfers to 180 μ l supernatants in flat 96 orifice plates and definite OD 405 in 96 hole microplate reader.

In addition, two control tube have been prepared to establish the OD reading of 100% and 0% Erab cracking.For 100% cracking, contain 40 μ l in the pipe and come the medium of cell of free GFP carrier transduction and 10 μ l through Mg²⁺What-EGTA washed contains 5 * 10⁷Individual erythrocytic Erab.In 37 ℃ revolution oscillator, behind the incubation, add 200 μ l icy waters so that make the OER cracking.For 0% cracking (blank), contain 90 μ l in the pipe and come the medium of cell of free GFP carrier transduction and 10 μ l through Mg²⁺What-EGTA washed contains 5 * 10⁷Individual erythrocytic Erab.In 37 ℃ revolution oscillator, behind the incubation, add 0.9% ice-cold salt solution of 150 μ l to prevent erythrocyte splitting.

The result

As previously mentioned, people's wild type fB and three-type-person's dominant fB part is made in the tissue culture medium (TCM) from biv vector institute transducer cell.Shown among Figure 17 that the culture supernatants and the wild type fB that contain fB1, fB2 and fB3 compete to suppress to substitute tiring of complement pathway hemolytic activity.In addition, data show: (1) can functionally be replaced endogenous people complement factor B and rebuild the alternative route hemolytic activity really in the human serum of exhausting factor B by people's wild type fB of vector encoded; (2) people fB1, fB2 and fB3 itself do not bring into play the function of similar wild type fB and do not rebuild the alternative route hemolytic activity in the human serum of exhausting factor B; (3) fB1 shown in do not show the remarkable inhibiting activity that blocking-up substitutes the complement pathway activity (it should be noted that fB1 shows and suppresses activity (data not shown) the ratio of higher fB1 and wild type fB 1: 6 time) under the ratio; (4) fB2 shows certain inhibition activity; (5) fB3 shows substituting effective inhibition of complement pathway activity.When mixing with wild type fB with 1: 1 ratio, fB3 has suppressed about 90% hemolytic activity.When 2: 1 ratio, fB3 has suppressed alternative route complement activity (Figure 17) fully.

Embodiment 16. mouse factor B mutant and people's factor B mutant substitute the inhibition of complement pathway activity to mouse

Material and method

Except fresh normal mouse serum available from Innovative Research or from the fresh extraction of mouse, basic identical described in equipment, reagent and buffer solution and the embodiment 15.

Except the biv vector of coding people wild type factor B and three-type-person's factor B mutant, also as having prepared the biv vector of encoding wild type mouse factor B and three kinds of mouse factor B mutant as described in the embodiment 15.People's factor B mutant is appointed ashfB 1, hfB2 and hfB3, and mouse factor B mutant is appointed as mfB1, mfB2 and mfB3.Then as carrier transduction ARPE cell as described in the usefulness as described in the embodiment 15 or Cf2Th cell so that produce mouse wild type factor B albumen and mouse mutant factor B albumen.Assessed the expression of wild-type mice factor B and three kinds of mouse factor B mutant by the Western engram analysis, and found that described expression is basic identical, for example by estimating in 2 times.

Substitute the test of complement pathway hemolytic activity

The mice serum of having exhausted complement factor B is not commercially available.We find that for for the alternative complement pathway in the mice serum activates, factor B is a rate-limiting factor.Therefore, by suitable dilute serum (is 4 times of dilutions for this research), can similarly test with the research among the embodiment 15.Mouse whole serum by will dilution and the medium of the cell that comes free biv vector transduction mix with the volume ratio of 1: 1 or 1: 2 to be set up competition and tests, described biv vector encode respectively GFP or mfB1, mfB2 or mfB3.Whether abreast, also used three-type-person's factor B mutant in this research can compete with endogenous wild-type mice factor B so that determine people's dominance factor B mutant; That is, determine whether can be in suitable mouse model evaluator factor B mutant.(in this, it should be noted that complement factor usually works (for example, seeing Horstmann etc., J Immunol (1985) 134:11401-4) in the species specificity mode and can not support from the complement activation in the serum of different plant species).

Set up the hemolytic activity reaction in the 14ml polystyrene round bottom pipe on ice.In every pipe, add said mixture and the fresh ice-cold Mg that makes of 50 μ l of 40 μ l²⁺-EGTA buffer solution.With the abundant vortex of described effectiveFisher Vortex Genie 2 devices.Then, in each pipe, add 10 μ l through Mg²⁺What-EGTA washed contains 5 * 10⁷Individual erythrocytic Erab, not vortex and gentle mixing the subsequently.Then with the revolution of 110rpm concussion with describedpipe incubation 1 hour in 39 ℃ of water-baths.Notice that these incubation conditions are optimized the test with mice serum, and they are different from the used incubation conditions of test of human serum.Then described pipe is put back on ice and added 0.9% ice-cold salt solution of 150 μ l with stopped reaction.After gentleness is mixed, with described effective Beckman centrifuge of closing brake centrifugal 5 minutes of 4 ℃, 2000rpm.Not disturbance precipitates and transfers to each supernatant of 180 μ l in flat 96 orifice plates and definite OD 405 in microplate reader.100% cracking and negative contrast (blank) sample are as foundation as described in the embodiment 15.

The result

Shown mouse and people's factor B mutant and endogenous mouse factor B competition tiring among Figure 18 with the alternative complement pathway in the inhibition mice serum.In addition, data show: (1) mfB3 in this research has suppressed that mouse substitutes complement pathway and mfB1 and mfB2 show less inhibition activity; (2) surprisingly, hfB2 and hfB3 have suppressed that mouse substitutes complement pathway and hfB1 does not suppress mouse and substitutes complement pathway in this research.This result make can be in mouse model body build-in test hfB3, and because the species specificity of the complement factor of expection does not reckon with this result.

Embodiment 17. mouse factor B mutant substitute the inhibition of complement pathway activity to the people

Material and method

Basic identical described in equipment, reagent and buffer solution and embodiment 15 and 16.

Basic identical described in the generation of the biv vector of encoding murine wild type factor B and dominant factor B mutant and embodiment 15 and 16.From basic identical described in the generation of the wild-type mice factor B of biv vector transducer cell and dominant factor B mutant and embodiment 15 and 16.Carry out the haemolysis test according to the method among the embodiment 15 with the serum of exhausting people's factor B.

The result

Embodiment 15 and 16 has shown that jointly people's dominant negativemutant factor B 2 and B3 can suppress the alternative complement pathway of people and mouse, and mouse dominant negativemutant factor B 3 can suppress the alternative complement pathway of mouse.Present embodiment is designed to determine whether mouse factor B mutant can suppress the people and substitute complement pathway.As shown in figure 19, data show in this research: (1) mfB3 has suppressed that the people substitutes complement pathway and (2) mfB1 and mfB2 do not demonstrate obvious inhibiting activity.Comprised that in addition wild type people factor B and people's factor B mutant are as the test contrast.

Embodiment 18. people's factor B mutant substitute the inhibition of complement pathway activity to pig

Material and method

Except fresh pig serum extracts from the miniature pig of Yucatan (Yucatan), basic identical described in equipment, reagent and buffer solution and the embodiment 15.

Basic identical described in the generation of the biv vector of coding people wild type factor B and people's dominant factor B mutant and the embodiment 15.Generation and embodiment 15 from wild type people's factor B of biv vector transducer cell and people's factor B mutant are described basic identical.

Except that the fresh pig blood serum substituting mice serum with dilution, it is described basic identical with embodiment 16 that pig substitutes the test of complement pathway hemolytic activity.Serum thinner ratio in this research is 1: 2,1: 4 and 1: 6.

The result

Because the size of pig eye is similar to human eye with vascular system thereby pig can be used as the larger animal phantom eye.We have determined thereby whether people's dominant factor B mutant can suppress pig and substitute complement pathway and might allow further simulate in the body in pig future.As shown in figure 20, the result shows that hfB3 has effectively suppressed pig and substituted complement pathway.HfB2 also shows and suppresses active, but hfB2 does not have hfB3 effective.

Prove as Figure 20 as if wild type people's factor B substitutes in the complement pathway pig has function.Particularly, along with the dilution of pig serum, the total titer of hemolytic activity reduces.Yet, when each dilution, add people's wild type complement factor B and improved hemolytic activity.Except the observation to people's wild type factor B biologically active in pig serum, but this discovery has supported that also described test is supposition to the restricted compound of pig and able person's complement-mediated hemolytic activity to factor B at least under these experiment conditions.Described result has further strengthened strategy of the present invention, and especially the blocking-up of factor B function or reduction can suppress to substitute complement pathway effectively.

The factor B cutting that embodiment 19.C3b relies on

Reagent and buffer solution and material and method

Purifying people Complement Factor D albumen and purifying people complement factor C3b albumen are available from Quidel (catalog number (Cat.No.) is respectively A409 and A413).The polyclonal antibody of anti-people's complement factor B is available from Quidel (catalog number (Cat.No.) A311).

VECTASTAIN ABC-Amp Western trace immunity detection reagent is available from VectorLaboratories (catalog number (Cat.No.) AK-6000).Phosphate buffer (PB) contains 8mM Na₂HPO₄With 2mM NaH₂PO₄, the pH value is 7.4.

Except the factor B albumen collected from the BIV transducer cell without any further concentration step using, basic identical described in the generation of the biv vector of coding people's factor B wild-type protein and three-type-person's dominant negative mutant albumen and the embodiment 15.

The factor B cutting test that C3b relies on

To add 25mM NaCl and 10mM MgCl with people's factor D (final concentration is 200ng/ml) and people's factor C3b (final concentration is 2000ng/ml) from wild type people's factor B albumen and three-type-person's factor B mutant protein (final concentration is about 500ng/ml) of transduction supernatant₂PB (PB+) in 37 ℃ of incubations 30 minutes.The amount of each composition is listed in the table below in 2 in the reactant mixture.In contrast, a group reaction is carried out under the C3b not having.

Factor D is that C3b relies on to the cutting of wild type factor B.When factor B combines with C3b, thereby factor B experience conformation change exposes the factor D cleavage site and allows factor D that factor B (93Kda) is cut into Bb (63Kda) and Ba (30Kda), thereby activates C3 convertase (C3bBb).Factor D can not take place when lacking C3b the cutting of factor B, or takes place with floor level.Should be noted that according to the Western engram analysis and be estimated as about 10 μ g/ml from the concentration of various people's factor B albumen by BIV coding of transducer cell supernatant.

After the reaction, 25 μ l reactant mixtures are mixed with reduction protein sample buffer solution,, cool off also and in 7.5%Tris-HCl PAGE gel (Bio-RAD), carry out the SDS-PAGE electrophoresis 90 ℃ of incubations 30 minutes.With described gel by semigel transferring system and trace to nitrocellulose filter.Then with the goat anti-people factor B polyclonal antibody (Quidel of described film with dilution in 1: 8000, catalog number (Cat.No.) 8000) surveys, then survey with the anti-goat biotinylation of rabbit IgG (H+L) antibody (Vector Laboratories, catalog number (Cat.No.) BA-5000) of dilution in 1: 5000.Detect with VECTASTAIN ABC-AmpWestern trace immunity detection reagent (Vector Laboratories).

Table 2

The result

Detected by the cutting of factor D to wild type people complement factor B and three kinds of factor B mutant.As shown in figure 21, when not having C3b, not to any effective cutting of wild type factor B or three kinds of factor B mutant.When having C3b, factor D has effectively been cut wild type factor B.It has also cut fB1 and fB2 to some extent.Yet, have little or no cutting to fB3, verified the effectively cutting of in the factor D cleavage site of fB3, introducing of BF D of sudden change.The importance of this discovery is that when not having this proteolysis cutting, C3 convertase (C3bBb) can not form and the activation of alternative complement pathway will be blocked or suppress.Should be noted that in Figure 21 because the sudden change of through engineering approaches in fB2 removed the N-glycosylation site, thereby from the molecular weight of the Bb of fB2 less than molecular weight from the Bb of wild type factor B or fB1.

Embodiment 20. factor B combine with C3b's

Reagent and buffer solution and material and method

Purifying people Complement Factor D albumen and purifying human complement c 3 b albumen are available from Quidel (catalog number (Cat.No.) is respectively A409 and A413).The polyclonal antibody of anti-people's complement factor B and C3 is available from Quidel (catalog number (Cat.No.) A311 and A413).The anti-goat IgG Fc fragment antibody of biotinylated rabbit is available from JacksonImmunoLaboratory (catalog number (Cat.No.) 305-065-046).

Phosphate buffer (PB) contains 8mM Na₂HPO₄, 2mM NaH₂PO₄, the pH value is 7.4.Lavation buffer solution contains 20mM Tris-HCL pH 8.0,0.15M NaCl, 1%NP-40 and 2mMEDTA.Phenylmethylsulfonyl fluoride (PMSF) is available from Sigma (catalog number (Cat.No.) P7626).Protease inhibitor cocktail is available from Roche (catalog number (Cat.No.) 11836170001).The albumin A sepharose 4B is available from Invitrogen (catalog number (Cat.No.) 15918-014).Normal rabbit igg is available from R﹠amp; D Systems (catalog number (Cat.No.) AB-105-C).VECTASTAIN ABC-Amp Western trace immunity detection reagent is available from VectorLaboratories.Except the factor B albumen collected from the BIV transducer cell without any further concentration step using, the generation of the biv vector of coding people's factor B wild-type protein and three-type-person's dominant negative mutant albumen is substantially as described in example 15 above.

The test that factor B combines with C3b

Wild type people's factor B and three-type-person's factor B mutant protein (final concentration is 500ng/ml) from the transduction supernatant are adding 25mM NaCl and 10mM MgCl with people's factor D (final concentration is about 200ng/ml) and people's factor C3b (final concentration is 2000ng/ml)₂PB (PB+) in 37 ℃ of incubations 30 minutes.The amount of each composition is listed in the table below in 3 in the reactant mixture.In contrast, a group reaction is carried out under the C3b not having.

Table 3

After cutting test, reaction tube is placed on ice and sample is carried out immunoprecipitation.The lavation buffer solution that contains PMSF and protease inhibitors mixing tab that each sample (500 μ l) and 1ml is ice-cold mixes.Add the normal rabbit iggs of 2 μ l (1mg/ml) then, described pipe is shaken at 4 ℃ added 100 μ l albumin A pearls in 1 hour then.After the mixing, described pipe was shaken 1 hour at 4 ℃ again, afterwards 14, centrifugal 5 minutes of 000rpm.Every portion of supernatant is transferred in the new pipe, is added the anti-people's complement factor B of 2 μ l polyclonal antibody, then with described pipe 4 ℃ of shaken over night.In every pipe, add then 100 μ l in ice-cold lavation buffer solution the albumin A pearl and described pipe shaken 1 hour at 4 ℃.Then with described pipe 14, centrifugal 1 minute of 000rpm, abandoning supernatant, and not disturbance precipitation, with described pearl with the ice-cold lavation buffer solution washing of having added PMSF and protease inhibitors mixing tab 3 times.Then that described pearl is resuspended and 14, centrifugal 1 minute of 000rpm.At last, use the 500mM MgCl of 1ml₂Buffer solution washs described pearl so that remove the albumen of any non-specific binding.14, the 000rpm rotation is after 1 minute, and abandoning supernatant adds 100 μ l reduction protein sample buffer solution and by vortex described pearl fully mixed.Then described pearl is heated to 95 ℃ and reaches 3 minutes to discharge albumen.By 14,000rpm removed described pearl in centrifugal 2 minutes and 90 μ l supernatants is transferred in the new pipe.

Each sample pipetting volume of 40 μ l is gone up and carried out SDS-PAGE and carry out the Western engram analysis then to 7.5%Tris-HCl PAGE gel (Bio-RAD).Gel is passed through semigel transferring system trace to nitrocellulose filter.Described film is surveyed with the anti-people's factor of the goat C3 polyclonal antibody of dilution in 1: 5000, and then with 1: 20, the anti-goat biotinylation of the rabbit IgG Fc fragment antibody of 000 dilution is surveyed.With VECTASTAIN ABC-Amp Western trace immunity detection reagent (Vector Laboratories) protein band is manifested.

The result

This experiment has detected the C3b binding characteristic of people's factor B mutant of comparing with wild type factor B.As shown in figure 22, carried out external combination test with C3b, factor B and factor D.Antiserum with the polyclone anti-factor B carries out immunoprecipitation to the reaction compound then, and under the sex change condition described compound is assessed with C3b polyclonal antibody probe by the Western analysis.The immunoprecipitation degree maximum of C3b and fB3 (Figure 22, the 4th swimming lane).Obviously littler with the amount of the C3b of fB2 immunoprecipitation, and with amount even littler (Figure 22, the 3rd, 5 and 6 swimming lanes) of the C3b of wild type factor B and fB1 immunoprecipitation.It should be noted that in this research owing to the compound of C3b and wild type factor B was survived with the known half life less than 2 minutes shortly, thereby the amount of the C3b of expection immunoprecipitation is less.Significantly very astonishing with the observation that combines of fB2 greater than C3b with combining of fB3 to C3b, this is to be identical because design changes in fB2 and fB3 in order to the amino acid that improves the C3b combination.Thereby the particular combinations of suddenling change among the fB3 so effectively causes still not knowing with the more firm reason that combines of C3b.The most important thing is that fB3 may be to be an explanation of the discovery of the alternative the most effective factor B dominant of the complement pathway body of inhibition to fB3 to the strong combination of C3b.Particularly, fB3 can cause the lasting isolation of C3b in the non-functional C3 convertase to the lasting combination of C3b.

Embodiment 21. factor D combine with the C3bB compound

Material and method

Except the anti-people's factor D of polyclone goat antiserum available from Quidel (R﹠amp; D Systems, catalog number (Cat.No.) AF-1824) in addition, basic identical described in reagent and buffer solution and the embodiment 20.

Basic identical described in the generation of the biv vector of coding people's wild type factor B albumen and three kinds of dominant people factor B albumen and the embodiment 15.

Except with anti-factor D antiserum the reaction compound being carried out immunoprecipitation and the Western trace being surveyed with the anti-people's factor B of polyclone goat antiserum, to factor D and combining of C3bB compound test with embodiment 20 in test described basic identical to fB with combining of C3b.

The result

Embodiment 19 and 20 shows that cutting that people fB3 can resistance factor D and it are tightr than wild type factor B, fB1 or fB2 with combining of C3b.Present embodiment is designed the binding characteristic of evaluation factor D and different C3bB compounds, and wherein each different C3bB compound has different people's dominant factor B analogs.There is and serves as the catalyzer that the factor B in the C3bB compound is cut into Ba and Bb in factor D with extremely low-level usually.Therefore, will expect all that at any time little factor D can combine with the C3bB compound.As expection, and as shown in figure 23, for factor B, fB1 and fB2, only a small amount of factor B and factor D co-precipitation.Interesting and very unexpectedly, with the fB3 of factor D co-precipitation much more (Figure 23, the 5th swimming lane).Thereby as if when C3bB and fB3 compound tense, factor D combines closely manyly with this compound.The mechanism of lasting combination is not so still known.The most important thing is that for why fB3 is more effective to suppressing to substitute complement pathway than fB1 or fB2, the factor D that continues is in conjunction with further explanation is provided.Particularly, after compound with C3b, thereby fB3 can obtain with factor D continue to combine isolation factors D.Can utilize and need factor D albumen to mediate many proteolysis cuttings on a small quantity because factor D only has, remove the effect that factor D will play effective this approach of blocking-up from substituting complement pathway.

Present embodiment and before this data of embodiment show that although all three kinds of dominant factor B partly play the function that reduction substitutes complement pathway, fB3 shows one's talent because of the most effective unexpectedly.In addition, mechanism studies show that the uniqueness of fB3 is tired may be because two attributes: 1) its with C3b combine closely and 2) the combining closely of itself and factor D.Thereby, entering when substituting complement pathway, fB3 has obtained not form with the continuing to combine and these two compositions are removed of C3b and factor D functional C3 convertase (or C5 convertase) from described approach.Final result is that the positive feedback of having blocked alternative complement pathway is amplified ring and effectively suppressed this approach.

Be not wishing to be bound by theory, the discovery that factor D and the compound of C3b and fB3 are combined closely may can't to cut fB3 relevant with factor D.This support will have the dominant factor D (for example, as described herein dominant factor D) of more weak proteinase activity through design as the extra mode that effectively suppresses to substitute complement pathway.Because it can't cut factor B, can estimate that described dominant factor D is more firm than wild type factor D with combining of C3bB compound base.The dominant factor D of institute's combination can prevent that wild type factor D from entering described compound, cutting factor B and activating C3 convertase.The structure of factor D has been characterized (Volanakis JE and NarayanaSVL, 1996, Protein Science 5:553-564) thereby made can design the more weak dominant body of proteinase activity.At last, dominant factor D part can be used separately or partly be used in combination with the dominant factor B.

Embodiment 22. anti-people's factor B monoclone antibodies substitute the inhibition of complement pathway to the people

Material and method

Except the mouse monoclonal antibody of anti-people's factor B available from the control mice IgG of Quidel (catalog number (Cat.No.) A227) and isotype coupling available from the eBioscience (catalog number (Cat.No.) 14-4714), basic identical described in equipment, reagent and buffer solution and the embodiment 15.

Substitute the test of complement pathway hemolytic activity

To handle rabbit erythrocyte with embodiment 15 described identical modes.Before hemolytic reaction, in each pipe with 12.5ng/ μ l purification of recombinant human factor B albumen and anti-people's factor B monoclone antibody with the mol ratio of 1: 0.5,1: 1,1: 2 and 1: 3 in the Mg-EGTA buffer solution (cumulative volume is 40 μ l) 4 ℃ of precincubation 30 minutes.In each pipe, be added in the 50 μ l serum of exhausting people's factor B of 25 times of dilutions in the ice-cold Mg-EGTA buffer solution of prepared fresh then.For over against looking after, in the serum of exhausting people's factor B of 25 times of dilutions of 50 μ l, prepared 500ng purifying people complement factor B albumen and volume has been increased to 90 μ l with the Mg2+-EGTA buffer solution.In each pipe, add 10 μ l then through Mg²⁺What-EGTA washed contains 5 * 10⁷Individual erythrocytic Erab.With reactant not vortex and gentle the mixing.Place 39 ℃ of water-baths 110rpm revolution concussion 40 minutes pipe.After 40 minutes, pipe is put back on ice and added 0.9% ice-cold salt solution of 150 μ l with stopped reaction.Mix then in closing the Beckman centrifuge of brake centrifugal 5 minutes of 4 ℃, 2000rpm each pipe is gentle.Not disturbance precipitates and each supernatant of 180 μ l is softly transferred in flat 96 orifice plates, and determines OD 405 in microplate reader.

The result

The present invention who is used to suppress to substitute complement pathway be to use on the other hand anti-this pathway components as binding molecules such as monoclone antibody (mAb) or its binding fragments, described pathway components includes but not limited to factor B, factor D, C3b, C3 convertase etc.Binding molecule directly can be carried (for example, being transported to retina) or expressed by gene transfer vector (as slow virus carrier etc.).Monoclone antibody can be used as fragment or single-chain antibody conveyings such as Fab fragments.Whether this experiment detects can effectively suppress alternative route as binding molecules such as monoclone antibodies.As shown in figure 24, the mAb of anti-people's factor B has closed alternative complement pathway activity basically at all four dosage of test.Because control mice IgG does not suppress or does not block pathway activities at similar dosage, thereby described inhibition has specificity.

Embodiment 23. anti-people's Complement Factor Ds substitute the inhibition of complement pathway activity to the people

Material and method

Except monoclonal anti-human factor D antibody available from Affinity Bioreagents (Golden, CO, catalog number (Cat.No.) GAU008-01-02) and the mouse of isotype coupling contrast IgG available from eBioscience (catalog number (Cat.No.) 14-4732) in addition, basic identical described in equipment, reagent and buffer solution and the embodiment 15.Except that the mAb that uses anti-factor D replaces the mAb of anti-factor B, substitute the active haemolysis test of complement pathway with basic identical described in the embodiment 22.

The result

The factor D representative substitutes the important component of complement pathway, therefore is the desirable target that suppresses described approach.This experiment is designed to show as approach as described in the suppressing in conjunction with albumen of anti-people's factor D such as mAb.As shown in table 4, the mAb of anti-factor D has suppressed alternative complement pathway hemolytic activity in dosage dependence mode.Because the control mice IgG of isotype coupling does not cause any inhibition, thereby described inhibition is that factor D is specific.

The anti-people's factor D of table 4. monoclone antibody substitutes the inhibition of complement pathway activity to the people

Sample	??OD??405nm	Sample	??OD??405nm
				Anti-hfD mAb 5ng+ purifying hfB albumen 0.5ug	??0.373	Contrast IgG 5ng+ purifying hfB albumen 0.5ug	??0.428
Anti-hfD mAb 10ng+ purifying hfB albumen 0.5ug	??0.432	Contrast IgG 10ng+ purifying hfB albumen 0.5ug	??0.361
				Anti-hfD mAb 20ng+ purifying hfB albumen 0.5ug	??0.369	Contrast IgG 20ng+ purifying hfB albumen 0.5ug	??0.441
Anti-hfD mAb 30ng+ purifying hfB albumen 0.5ug	??0.235	Contrast IgG 30ng+ purifying hfB albumen 0.5ug	??0.392
				Anti-hfD mAb 40ng+ purifying hfB albumen 0.5ug	??0.227	Contrast IgG 40ng+ purifying hfB albumen 0.5ug	??0.426
Anti-hfD mAb 50ng+ purifying hfB albumen 0.5ug	??0.165	Contrast IgG 50ng+ purifying hfB albumen 0.5ug	??0.430
				Anti-hfD mAb 60ng+ purifying hfB albumen 0.5ug	??0.126	Contrast IgG 60ng+ purifying hfB albumen 0.5ug	??0.416
Anti-hfD mAb 70ng+ purifying hfB albumen 0.5ug	??0.103	Contrast IgG 70ng+ purifying hfB albumen 0.5ug	??0.395
				Anti-hfD mAb 80ng+ purifying hfB albumen 0.5ug	??0.078	Contrast IgG 80ng+ purifying hfB albumen 0.5ug	??0.387
Anti-hfD mAb 90ng+ purifying hfB albumen 0.5ug	??0.058	Contrast IgG 90ng+ purifying hfB albumen 0.5ug	??0.347
				Anti-hfD mAb 100ng+ purifying hfB albumen 0.5ug	??0.061	Contrast IgG 100ng+ purifying hfB albumen 0.5ug	??0.397
Anti-hfD mAb150ng+ purifying hfB albumen 0.5ug	??0.035	Contrast IgG 150ng+ purifying hfB albumen 0.5ug	??0.426
				Anti-hfD mAb 200ng+ purifying hfB albumen 0.5ug	??0.029	Contrast IgG 200ng+ purifying hfB albumen 0.5ug	??0.347
Anti-hfD mAb 400ng+ purifying hfB albumen 0.5ug	??0.022	Contrast IgG 400ng+ purifying hfB albumen 0.5ug	??0.506
				Anti-hfD mAb 800ng+ purifying hfB albumen 0.5ug	??0.015	Contrast IgG 800ng+ purifying hfB albumen 0.5ug	??0.538
Anti-hfD mAb1600ng+ purifying hfB albumen 0.5ug	??0.014	Contrast IgG 1600ng+ purifying hfB albumen 0.5ug	??0.618
				Anti-hfD mAb 2400ng+ purifying hfB albumen 0.5ug	??0.049	Contrast IgG 2400ng+ purifying hfB albumen 0.5ug	??0.588
Over against shining (100% cracking)	??1.453
				Negative contrast	??0
Purifying hfB albumen 0.5ug	??0.399

By the haemolysis testing evaluation activity of alternative complement pathway.Measure relative hemolytic activity by the amount that is discharged into the haemoglobin in the supernatant behind the rabbit reticulocyte lysate.100% cracking over against photograph, the RBC of cracking in water; Purifying hfB albumen has replenished the human serum of exhausting factor B of the purifying people factor B albumen of 500ng; Negative contrast is with red blood cell incubation (no erythrocyte splitting) in isotonic saline solution; Replenished the purifying people factor B albumen of 500ng and the mAb of anti-people's factor D (5ng～2400ng) or the control mice IgG (human serum of exhausting factor B of the mixture of 5ng～2400ng).

Embodiment 24. rabbit anti-factor B monoclone antibodies are to the inhibition of the alternative complement pathway of people and mouse

Material and method

Except the mAb that adopts rabbit anti-factor B described in the present embodiment replaced the mAb of mouse anti factor B, it was described basic identical with embodiment 22 to substitute the test of complement pathway haemolysis.

Increase the antibody that generates at people's factor B thereby we have generated mAb in rabbit and can set up the animal mould model of mouse with the possibility of mouse factor B cross reaction is feasible.People's factor B of purifying also is used as the antigen of immunize rabbit available from Quidel (catalog number (Cat.No.) A408).Antibody is by GenesisBiotech, and Inc. (Taiwan) generates according to its standard method.With two rabbit immunity, splenocyte and fusion partners are merged, identify hybridoma then, and screen the secretion of mAb by ELISA.By embodiment 22 described haemolysis tests 20 portions of hybridoma supernatants are further analyzed the ability that its blocking-up substitutes complement pathway.The hybridoma medium (from 1ml hybridoma medium) of freeze-drying is dissolved among 1 * PBS of the everypipe 250 μ l of total amount.To store with aliquot at-80 ℃ through the resuspended hybridoma solution of PBS.

Before hemolytic reaction, with people's factor B albumen of each the hybridoma medium of 20 μ l and 500ng purifying in the Mg-EGTA buffer solution with the total amount of 40 μ l 4 ℃ of precincubation 30 minutes.For antibody over against photograph, with 500ng purifying people factor B albumen and 400ng or the anti-people's factor B of 800ng monoclone antibody (Quidel) in the Mg-EGTA buffer solution with 40 μ l 4 ℃ of precincubation 30 minutes.Then, in each pipe, dilute 25 times the serum of exhausting people's factor B in the ice-cold Mg-EGTA buffer solution of interpolation 50 μ l in prepared fresh.For over against looking after, in the serum of exhausting people's factor B of the described 25 times of dilutions of 50 μ l, prepare 500ng purifying people's complement factor B albumen and use Mg²⁺-EGTA buffer solution increases to 90 μ l with volume.In each pipe, add 10 μ l then through Mg²⁺What-EGTA washed contains 5 * 10⁷Individual erythrocytic Erab.With reactant not vortex and gentle the mixing.Place 37 ℃ of water-baths 110rpm revolution vibration 40 minutes described pipe.After 40 minutes, described pipe is put back on ice and added ice-cold 0.9% salt solution of 150 μ l with stopped reaction.Mix then in closing the Beckman centrifuge of brake centrifugal 5 minutes of 4 ℃, 2000rpm each pipe is gentle.Not disturbance precipitates and each supernatant of 180 μ l is softly transferred in flat 96 orifice plates, and determines OD 405 in microplate reader.

In order to detect the inhibition of described 20 parts of rabbit mAb, except that the mouse whole serum with 4 times of dilutions replaces having exhausted the human serum of factor B, carried out identical experiment to the alternative complement pathway activity of mouse.

The result

The factor B of people and mouse is about 83% homology at amino acid levels.It is desirable to produce the mAb that not only can be used for setting up the animal model of rodent but also can be used for the human treatment.Shown in table 5 and table 6,20 positive rabbit hybridomas have been produced.The alternative complement pathway that has suppressed people's (table 5) and mouse (table 6) by the monoclone antibody of some generation in these clone body.These positive hybridomas can be as the source that produces single-chain antibody, and described single-chain antibody can be by humanization as for example human treatment.This class single-chain antibody can be carried by protein injection or by the carrier of the described single-chain antibody of encoding.Select as another kind, can carry, for example be used for the treatment of purposes with rabbit mAb humanization and as the Fab fragment or as intact proteins.Strategy in the present embodiment can be used for producing the rabbit monoclonal antibodies that has at the treatment effectiveness of other alternative complement pathway key component (for example, factor D, C3b or C3 convertase).

Table 5: the mAb with anti-hfB suppresses complement activity

Table 6: the mAb with anti-hfB suppresses to substitute the complement pathway activity

Embodiment 25. purifying people fB3 albumen suppress to substitute complement pathway

Some embodiment of the present invention comprises that conveying can suppress or block the albumen as approach such as alternative complement pathways.For example, present embodiment has been described use fB3 albumen and has for example been suppressed to substitute complement pathway.

As described herein, fB3 albumen can be made in mammalian cell, bacterium, yeast, insect or other biology of living.This research be designed to determine we whether can be from medium purifying fB3 albumen and still keep its biologically active.Biv vector transduction Cf2Th cell (seeing embodiment 15) with coding people fB3.The cell of transduction is maintained in the DMEM medium that contains 2%FBS.Transduceed back 72 hours, thus collecting cell medium and filter the scavenger-cell chip through 0.2 μ m sterilizing filter.Cell culture medium application of sample after removing there is anti-people's factor B monoclone antibody (R﹠amp to coupling; D Systems, catalog number (Cat.No.) MAB2739) on the affinity column (PIERCE, catalog number (Cat.No.) 44894).Wash described post and according to operation instructions with the sample wash-out.Assess the wash-out flow point by SDS-PAGE with silver dyeing.And detect described flow point so that checking albumen identity by the Western engram analysis.Shown in Figure 25 A and 25B, described affinity column has produced the pure relatively fB3 of desired size.Figure 25 A has shown the silver dyeing of people'sfactor B 3 albumen of affinity purification: the 1st swimming lane, molecular weight marker; The 2nd swimming lane is from the elution samples of first flow point; The 3rd swimming lane is from the elution samples of the combination of second flow point and the 3rd flow point.Figure 25 B has shown identical among the Western engram analysis of people'sfactor B 3 albumen and lane assignment and the figure A.

In addition, do not have tangible catabolite, this shows 3 pairs of described purge process well-tolerated of mutant factor B.The extra band identical with the BSA molecular dimension (Figure 25 A, the 2nd and the 3rd swimming lane) arranged at about 65Kda place.

For whether themutant factor B 3 of determining purifying has biologically active, as described in embodiment 15, carried out alternative complement pathway haemolysis test.As shown in figure 26, the people fB3 albumen of affinity purification has effectively suppressed the people and has substituted complement pathway, thereby shows that described purge process does not cause any obvious damage to the biological function of described albumen.Also can come purifying fB3 albumen by other method or with the combination of other method, for example, ion-exchange chromatography, size exclusion chromatography, ammonium sulfate precipitation, HPLC.

The cell-line of embodiment 26. constitutive expression people fB3

In order more easily to make and purifying fB3, we have generated the cell-line of constitutive expression fB3 in the suspension culture base of serum-free.Particularly, use the carrier transduction 293Freestyle cell (Invitrogen) (for example, seeing embodiment 15) of coding fB3.Cell is grown in the suspension cell culture mode in 293Freestyle serum free medium (Invitrogen).Tissue culture medium (TCM) is carried out analyzing as embodiment 25 described SDS-PAGE and Western.Discovery fB3 albumen is expressed justacrine (data not shown) in tissue culture medium (TCM).By test as embodiment 25 described haemolysis the biologically active of assessing fB3 and as described in fB3 demonstrate biologically active (data not shown).Also from Chinese hamster ovary celI and Cf2Th cellular expression fB3 albumen.

FB3 albumen as herein described or further modification of other albumen can be tired with increase, for example, be used for the treatment of purpose.For example, can be with albumen and polyethylene glycol conjugation (PEGization) so that increase half life in its body; Albumen can be carried by the device that discharges this albumen when implanting; Albumen can be made as fusion so as to increase its tire or body in half life; Albumen can be mixed with carrier so that increase in its body and distribute; Can be with albumen by expressing the cell delivery of described albumen; And/or can be with the albumen brachymemma to prepare the variant that still keep its biological function.

The biv vector of embodiment 27. coding people FB3 suppresses the retina inflammation in vivo

Present embodiment has been assessed the biv vector of coding people fB3 in the mouse model of retina Stimulated Light damage.In this extreme inflammatory model, thereby in a few hours, cause substituting the quick active of complement pathway with the laser burn retina.

Under study for action, every mouse is accepted the carrier of coding fB3 or the subretinal injection of the genetically modified carrier of not encoding in retinal periphery.After 2 weeks, near the foveal region of retina part, carry outlaser burn 3 times.After 20 hours, collect retina and to the sediments dyeing of complement factor C3 or membrane attack complex (MAC), complement factor C3 and membrane attack complex are the signs of complement activation.

For size and the position that shows vector injection, independent one group of cohort mouse has been accepted the injection of the carrier of coding GFP.This cohort is damaged without Stimulated Light.

Method

By prepare and concentrated the carrier or the genetically modified carrier (empty carrier) of not encoding of coding GFP, people fB3 as embodiment 15 described anion-exchange chromatographies.Store buffer liquid is to have added 1mMMgCl₂, 2.5mM KCl and 0.1%BSA PBS.Carrier stores for future use at-80 ℃.

Following the GFP carrier is carried out titration:, inoculate 2 * 10 among the DMEM at 3ml in every hole of 6 orifice plates at the 1st day⁵Individual Cf2TH cell is supplemented with 2mM glutamine, Pen/Strep and 10%FBS (DMEM fully) in described DMEM.With described cell in 37 ℃ at 8.5%CO₂In be incubated overnight.Next day is with the complete DMEM replacement medium that is added with 8 μ g/ml cohesion amine of 1.5ml.In every hole, add carrier (0.2 μ l or 1 μ l) and with plate incubation 15 hours～18 hours.Fresh complete DMEM with 3ml replaces medium then.After 49 hours～52 hours, pair cell carries out flow cytometry, and assesses the percentage (promptly than stronger without the fluorescence of the cell of transduceing) of fluorescigenic cell.Recently determine titre with mathematical method from carrier input quantity, hole inner cell number and fluorecyte percentage.

In order to determine the titre of hfB3 carrier and empty carrier, use as above transducer cell of whole three kinds of carriers.Yet, the cell after the transduction do not carried out flow cytometry but carry out PCR in real time so that determine its carrier DNA copy number.DNA by conventional method from described cell preparation.Can the increase PCR primer in RPE district of described biv vector of design:

Probe: 5 '-FAM-ACACCACCATCCCTCCGCATCCGA-BHQ-1-3 ' (SEQID NO:24)

Sense primer: 5 '-TGGGTTTGTGGTAGTAAATGACAC-3 ' (SEQ IDNO:25)

Antisense primer: 5 '-TGGTTCACGAGCGTTGTAGC-3 ' (SEQ ID NO:26)

(IQ5 Multicolor Real-Time PCRDetection system BioRad) carries out pcr amplification with following condition: 100nM probe, 600nM sense primer, 600nM antisense primer and 1 * SuperMix (BioRad) with IQ5 multicolor real time PCR detection system.Reactant then carried out 40 circulations in 3 minutes at 95 ℃ of incubations: 95 ℃ 10 seconds, 55 ℃ 30 seconds.By relatively the hFB3 carrier and the DNA copy number of empty carrier and the DNA copy number of GFP carrier have determined that with transduced unit (tu)/ml be the hFB3 carrier of unit and the titre of empty carrier.

Studying for this, is that the titre of unit is as follows with tu/ml: GFP carrier, 9 * 10⁷The hfB3 carrier, 3.2 * 10⁷And empty carrier, 7.3 * 10⁷

The subretinal injection method is as follows.Every mouse (C57BL/6) is accepted ketamine (100 μ g/gm) by intramuscular injection.Regulate dosage to obtain deep layer anesthesia.Before carrying out described method, with local 0.55 keracaine eye is handled immediately.Under anesthesia, leniently the people is for making eyes outstanding.Pass the just little otch to the corneal limbus of sclera with the cutting edge manufacturing of No. 30 pins.Towards eye back extremely tangentially insert No. 33 blunt nosed pins and place retina and layer of retina,pigment epithelium between.Inject 0.5 μ l～1 μ l carrier suspension, lift from the surface and confirm inject successfully by observing injection site place retina (it should be noted that the liquid of injecting in 1 day is absorbed and retina adhere to again).Take out syringe needle and keep pressure in case non-return leaks (back leakage).Observing described animal sends it in cage back to then until reviving and can walking about.

Vector injection is collected retina from the animal of having accepted the GFP carrier after 2 weeks; The smooth sample of preparation retina; And observe GFP and express (Figure 27 A and 27B).

Vector injection is after 2 weeks, to accepting the following laser burn that carries out of eye of hfB3 carrier and empty carrier.Employing is carried out laser light with fixed attention with hand-held cover glass as the diode laser (810nm is from the OcuLight Six of IRISMedical) and the Zeiss slit-lamp system of haptic lens.Laser parameter is set at: intensity 100mW, 75 microns in some footpath, 0.1 second duration and pulse.In every eye, carry out burning for 3 times (at 3 o'clock, 12 o'clock and 9 o ' clock positions) and at every apart from 2～3 disc diameters of optic nerve.Be tested and appraised at the bubble that burns site and form the success (being breaking of Bruch's membrane) of confirming burning method.In advance these laser parameters have been carried out optimization excessive infringement has not been provided so that as one man provide success to burn; It is hemorrhage promptly to burn the site.

After the damage from laser 20 hours, collect retina and the complement factor C3 that burns site or the sediments of MAC are dyeed.Put to death animal with excessive ketamine and xylazine, and pluck eye immediately.After in 4℃ 4% paraformaldehyde, fixedly spending the night, under the Nikon disecting microscope, careful dissection of every eye removed anterior chamber of eye and vitreum.From RPE layer careful separation, the remaining RPE-choroid of radial cuts-sclera complex is to form smooth sample then with neural retina.Described complex is carried out immunohistochemical staining.

To the sedimental Cell immunohistochemical staining method of C3 following (similarly carrying out the MAC colouring method).With the smooth sample of each RPE-choroid-sclera complex with TBS (the Tris buffer saline: Tris/Tris-HCl 25mM, NaCl 0.13M, KCl 0.0027M, pH 7.4 ± 0.13; FisherScientific, catalog number (Cat.No.) BP2471-100) wash each 5 minutes for several times to remove paraformaldehyde.The 2%BSA and the 1%Triton X 100 that are used in then among the TBS sealed 1 hour each complex.With TBS that describedcomplex washing 3 times is each 5 minutes subsequently.It is then each 5 minutes withTBS washing 3 times room temperature sealing 2 hours to described complex to be used in 10% normal rabbit serum (NRS) among the TBS and 1%Triton then.Then with described complex be incubated overnight at 4 ℃ to be diluted in one among the TBS that is added with 10%NRS and 0.5%Triton anti-(from the anti-people C3 of the polyclone goat of Calbiochem, catalog number (Cat.No.) 204869) at 1: 100.With the TBS washing that is added with 0.2%Tween 20 3 times each 10 minutes, then carried out onetime 10 minutes washing with TBS with described complex inferior morning.Then with described complex with to be diluted in two among the TBS anti-(coupling the anti-goat IgG of rabbit of Alexa fluor 594,, catalog number (Cat.No.) A-11080) at 1: 300room temperature incubation 2 hours from Invitrogen.Described complex with the TBS washing of having added 0.2%Tween 20 3 times each 10 minutes, was then washed 5 minutes with TBS.Described complex is locked on the slide under the cover glass with the Vectashield mounting medium.Detect described complex by fluorescence microscope then.Contrast dyeing has been omitted one and has been resisted.Although it should be noted that described one anti-generation at people C3, it also dyes to mouse C3.

The result

Figure 27 A and 27B have shown the GFP dyeing from two parts of smooth samples of representative retina.In each situation, inject the retina peripheral region of having transduceed.Figure 27 C and 27D have shown the eye of the empty carrier treatment of using by oneself and have dyeed with the C3 of the representative laser burn of the eye of the vehicle treatment of coding hfB3.The area of the C3 dyeing in the eye of empty carrier treatment and intensity are significantly greater than the C3 dyeing in the eye of hfB3 vehicle treatment.Result's similar to the C3 coloration result (data not shown) of MAC dyeing.

Conclusion

The carrier of coding hfB3 has suppressed complement activation effectively in this damage from laser model.Described result is former thereby make the people with deep impression especially for two.At first, this is very extreme complement activation model, and wherein said complement activation is caused by acute burn.Secondly, just obtained usefulness with the minute quantity transducer cell that is positioned at away from damage from laser point.The usefulness of inflammation of eye section animal model that should be relevant has indicated the usefulness in human disease treatment.

The assessment that the people FB3 albumen of the method for complement activation is carried is blocked in 28. pairs of conducts of embodiment in vivo

This experiment is designed to show that hfB3 albumen can suppress complement activation when carrying by direct intraocular injection.Research among this research and the last embodiment is carried out in a similar manner.In this situation, with the people fB3 albumen people fB3 albumen of the method in embodiment 25 or 26 preparation (for example, by) by in the vitreum and/or subretinal injection use.The difference of intravitreal injection method and subretinal injection method only is to place syringe needle in the vitreum rather than under the retina.In each case, volume injected is about 1 μ l and the concentration range of fB3 is 1ng/ μ l～20 μ g/ μ l.With not injecting the contrast eye with the preparation of hfB3.Carry out injecting at once behind the laser burn.Damage from laser is collected retina and is compared to C3 or MAC dyeing and with the injection with preparation only after 20 hours.

Embodiment 29. carriers concentrate and purification process: use the amplification of Sartobind SingleSep Mini Q membrane adsorbent capsule

It below is the detailed method that is used to amplify based on the purge process of the slow virus carrier of BIV.

Reagent and material

A.Benzonase, ultrapure, Sigma catalog number (Cat.No.) E8263 or equal product

B.1 * PBS pH 7.4, Invitrogen, catalog number (Cat.No.) 10010-023 or equal product

C.10 * PBS pH 7.4, Invitrogen, catalog number (Cat.No.) 70011-044 or equal product

D. distilled water is aseptic, no DNA enzyme, RNA enzyme, Invitrogen catalog number (Cat.No.) 10977015 or equal product

E.5M NaCl, Cambrex catalog number (Cat.No.) 51202 or equal product

F.Sartobind SingleSep Mini Q, Sartorius catalog number (Cat.No.) 92IEXQ42D4-OO

G.Vivaspin 20,1 1,000,000 MWCO, Sartorius catalog number (Cat.No.) VS2061

H.Vivaspin 20 diafiltration cup, Sartorius catalog number (Cat.No.) VSA005

The i.Nalgene pipe, 180PVC, FDA/USPVI, 1/8 " ID * 1/4 " OD s 1/16 " wall or equal product

J.EGTA, BioChemika Ultra＞99%, Sigma catalog number (Cat.No.) 03778 or equal product

K. storage bottle is disposable, various sizes, Corning or equal product

L. centrifuge tube, 250ml, Corning

M. centrifuge tube, 50ml, Falcon

N.MF75aPES 0.2 μ m filter element, 1 liter, Nalgene production number 567-0020 or equal product

O.0.2 μ m PES, 26mm, aseptic injection filter, Corning production number 431229 or equal product

P. syringe, 3ml, Becton-Dickinson, BD production number 309585 or equal product

Q. syringe, 60ml, Becton-Dickinson, BD production number 301627 or equal product

R. serological pipe, various sizes, aseptic, independent packaging

S. ring stand and folder

T. peristaltic pump and pump head, Masterflex L/S or equal product

U. centrifuge, rotor are Beckman Allegra 6KR or the equal product of GH3.8

Sample preparation

A. from the cell culture medium that contains carrier of 1250ml, described carrier is by being prepared (unconcentrated carrier) asembodiment 1 described calcium phosphate plasmid transfection.

B. the described carrier branch that do not concentrate is installed in the 250ml centrifuge tube.

C. it is in the Beckman Allegra 6KR centrifuge of GH3.8 centrifugal about 10 minutes at rotor, so that remove cell debris with 2800RPM.

D. 600ml is not concentrated carrier and put into twoCorning 1 litre flasks.

E. (ultimate density is 50 units/ml) to add the 500mM EGTA (10mM ultimate density) of pH 8.0 of 12ml and 30,000 Benzonase of unit in each bottle.37 ℃ of incubations 30 minutes～40 minutes.

F. the carrier that do not concentrate with every part of 600ml filters by Nalgene 0.2 μ m PES filter element.

Last sample

A. with cold sample-loading buffer with described 1, the dilution of 200ml carrier is 1: 1.Place on ice.Sample-loading buffer (2 * PBS, 1.0M NaCl): *

240.0ml?10×PBS，pH?7.4

165.6ml?5M?NaCl

794.4ml there is not the sterile water (cold) of DNA enzyme and RNA enzyme

1,200ml

B. according to operation instruction pipe is placed in the head of peristaltic pump.

C. will manage end and put into the container that contains 1 * PBS.Be connected with SingleSep Mini Q and remove air in pipe and the capsule unit with 1 * PBS of about 250ml.Guarantee all air are removed from Sartobind Mini Q capsule.

D. the end of feed pipe is carefully put into and do not concentrated carrier solution.

E. make sample solution pass through described unit with about 12ml/ minute speed.Continuation remains minimum sample in feed containers.Not with in the air intake pipe.

Washing

A. feed pipe is carefully taken out and puts into cold lavation buffer solution from shuttle.Do not make air enter SingleSep Mini Q unit.

B. with the speed washing SingleSep MiniQ of about 200ml lavation buffer solution with 12ml/ minute.

Lavation buffer solution (1 * PBS, 500mM NaCl):

40.0ml?10×PBS，pH?7.4

27.6ml?5M?NaCl

332.4ml there is not the sterile water (cold) of DNA enzyme and RNA enzyme

Be total to 400ml

Wash-out

A. in the 60ml syringe, charge into the cold elution buffer of 40ml

Elution buffer (1 * PBS, 1.3M NaCl):

20.0ml?10×PBS，pH?7.4

45.8ml?5M?NaCl

134.2ml there is not the sterile water (cold) of DNA enzyme and RNA enzyme

Be total to 200ml

B. be connected to SingleSep Mini Q, guarantee not introduce air.

C. keep described unit vertical, the utmost point is dropwise pushed lentamente by 9ml elution buffer.Abandon.

D. make it to leavestandstill 10 minutes～15 minutes.

E. new 50ml centrifuge tube is placed under the SingleSep Mini Q and the about 20ml of collection.

Further concentrate and diafiltration

A. the Beckman Allegra 6KR that with rotor is GH3.8 is chilled to 10 ℃ in advance.

B. the carrier of wash-out is carefully placed in two Vivaspin Unit 20 (1 * 10⁶MWCO).At rotor is among the Beckman Allegra 6KR of GH3.8 with 2200RPM centrifugal 20 minutes, checks volume and centrifugal again up to residue about 2ml (liquid is completely contained in device " V " shape district)

C. the diafiltration cup is put into the unit, shift the end onto under the cup.

D. in the diafiltration cup, add the 12ml diafiltration buffer and (be supplemented with 1mM MgCl₂With 1 * PBS) of 2.5mMKCl.

E. rotor be among the Beckman Allegra 6KR of GH3.8 with Vivaspin 20 centrifugal 15 minutes of 10 ℃, 2200RPM.

F. the carrier bulk of checking residual volume and rotating as required in each of two Vivaspin 20 diafiltration module again is about 750 μ l.

G. take the diafiltration cup away.Collect carrier by drawing (about 10 times) for several times up and down.

H. merging two parts of diafiltration things is the concentrated carrier of 1.5ml to obtain final volume.

I. by 0.2 μ M PES injection filter aseptic filtration.

J. be stored in-80 ℃.

The titre of the 2ml carrier products that this method produces routinely is to be low to moderate medium 10⁸In the tu/ml scope, plasmid DNA is polluted less than 50pg/ml, and host cell DNA pollutes less than 4pg/ml and Benzonase pollution detection less than (less than 0.1ng/ml).Described method is not removed BSA fully.When concentrating the carrier starting material and contain 10%FBS, the BSA level in the carrier of concentrating is 7 μ g/ml～21 μ g/ml (BSA ELISA from Cygnus Technologies, Southport, NC, catalog number (Cat.No.) F030).This method further can be amplified to satisfy and make requirement.

Embodiment 30. the carrier of embodiment 29 concentrate and purification process in add the size exclusion chromatography purification step

It below is the method that is further purified the carrier of embodiment 29 and reduces the BSA level.After the diafiltration steps of embodiment 29, but before aseptic filtration, the carrier through concentrating is added to SephacrylS 500-HR post (Sigma).

Load empty 20ml Econo-Pac chromatographic column (BioRad) with Sephacryl S 500-HR under gravity, bed volume is 20ml.

A. use the store buffer liquid of 5 volumes (to be supplemented with 2.5mM KCl and 1.0mM MgCl₂PBS) carry out balance.

B. the concentrated carrier of about 1.5ml is carefully added to the Sephacryl top and allows described carrier enter Sephracryl under gravity.

C. add extra store buffer liquid at capital, make the post current drainage under gravity and collect the flow point of 0.5ml～1.0ml.Carrier sees in the elution volume of 6.5ml～9.5ml.

D. merge the flow point contain carrier, aseptic filtration and-80 ℃ of storages.

Compare with the diafiltration thing that applies on post, the carrier titre in the eluate is diluted, and 2 times and productive rate are about 90%.In research shown in Figure 28, not concentrating the carrier starting material is in containing the tissue culture medium (TCM) of 2%FBS (embodiment 31 sees below).The BSA content of the diafiltration thing that applies on post is 311ng/ml.As shown in Figure 9, the adding of this size exclusion chromatography step separates BSA effectively with carrier.This method further can be amplified to satisfy and make requirement.

Embodiment 31. collects carrier in the defined medium of low serum or serum-free

Embodiment 1 described upstream process relates in calcium phosphate mediates four plasmid transfections to 293,293T or 293FT cell, collects the medium that contains carrier then by adding butyrate change medium thereafter.The foregoing description great majority have adopted the tissue culture medium (TCM) that contains 10%FBS.The inventor finds, might collect carrier in FBS medium still less, or uses and be specified to the medium of branch fully and remove all FBS and lost by any titre.In the present embodiment, with all cells cover plant in the DMEM that has added 10%FBS.Next day, medium is changed to the DMEM that has added 10%FBS or 2%FBS.Transfectional cell after 3 hours.After the transfection 18 hours, the DMEM of medium has been changed to the interpolation that respectively contains 5mM sodium butyrate 10%FBS or 2%FBS, or medium is changed to the CD CHO medium (Invitrogen) of not being with any FBS but containing the 5mM sodium butyrate.After 24 hours, collect medium and determine the carrier titre.Result shown in the table 7 shows the carrier that can carry out calcium phosphate transfection and can obtain high titre in 2%FBS in the medium of determining composition fully of serum-free.The inventor also determines and the upstream process of transfection and collection carrier can be amplified in Nunc cell factory (Nunc Cell Factories) and the approaching output (titre is in 2 times) that is obtained in the tissue culture ware of output.

Table 7

Transfection media	Collect medium	Titre
			??DMEM+10％FBS	??DMEM+10％FBS	??4x106tu/ml
??DMEM+10％FBS	??DMEM+2％FBS	??4x106tu/ml
			??DMEM+10％FBS	CD CHO medium (no FBS)	??5x106tu/ml
??DMEM+2％FBS	CD CHO medium (no FBS)	??7x106tu/ml

The carrier of embodiment 32.embodiment 1 and embodiment 29～31 concentrates the combination with purification technique

As described in theembodiment 1 with 293FT cell (Invitrogen) cover plant in the DMEM that adds 10%FBS.Next day, in every ware, medium is changed to the DMEM that adds 2%FBS.As described inembodiment 1, carry out four kinds of calcium phosphate transfections that produce the plasmid of carrier.After 18 hours medium is changed to without any FBS but contains the CD CHO medium (Invitrogen) of 5mM sodium butyrate.After 24 hours, collect medium and carry out concentrating and purification process among the embodiment 29 with the SEC combination of embodiment 30.Figure 28 has shown from the carrier titre in each flow point of Sephacryl post.In any flow point, do not detect BSA from the Sephacryl post.The detection limit of BSAELISA (Cygnus Technologies, Southport, NC catalog number (Cat.No.) F030) is 500pg/ml.This scalable method provides the carrier of high-purity, high titre.

Embodiment 33.AKTA purifying instrument purifying people fB3

By be used to catch, the combination of anion-exchange chromatography (IEXQ), hydrophobic interaction chromatograph (HIC) and the size exclusion chromatography (SEC) of intermediate purification and purification step carries out purifying to solubility secretion fB3 from the culture supernatant of cell (for example, Chinese hamster ovary celI or 293 cells).The cell culture supernatant that will contain fB3 concentrates 40 times～50 times and with 50mMTris-HCl the pH value is adjusted to 9.0 on the milipore filter that 30kDa holds back, then application of sample (HiTrapCapto Q, GE Healthcare) to the anion-exchange column of pre-filling.Described post carries out balance by the buffer solution (buffer A, electrical conductivity 5mS/cm) of the 50mM Tris-HCl that contains pH 9.0 in advance and detects in 280nm monitoring stream fluid by UV under the linear flow rate of 60ml/h.Not behind the bond material wash-out, by using nonlinear gradient progressively the electrical conductivity of flowing phase to be increased to 16mS/cm (10% buffer B, 10CV), 34mS/cm (27% buffer B, 10CV), 54mS/cm (50% buffer B, 5CV) and 101mS/cm (100% buffer B, thus 10CV) to mix the material that wash-out keeps with buffer B.By the SDS-PAGE under the reducing condition with by the flow point of elisa assay from anion-exchange column.Detected most of fB3 in the material of wash-out in second step (27% buffer B) between electrical conductivity 18mS/cm～30mS/cm, it contains total fB3 of 80% the input of having an appointment.These flow points contain the principal item (total application of sample material of 50%～70%) of the 93kDa that has an appointment, and this is corresponding to the fB3 that reduces fully.Yet, also may have other albumen impurity in these flow points.In case of necessity, can use the haemolysis test and whether keep its dominant activity so that determine the fB3 of purifying under this condition as functional test.Positive findings will show that the fB3 that increases dosage in this purification step can prevent the haemolysis that substitutes the mediation of complement activation approach, shows through the fB3 of anion exchange purifying to have kept its dominant activity with respect to wild type fB.

In order to help to remove deproteinized impurity, can adopt intermediate steps.This intermediate steps can be used hydrophobic interaction chromatograph, hydrophobic interaction chromatograph mainly based on the difference of the surface hydrophobic of albumen with their purifying with separate.To merge from the main flow point that contains fB3 of anion-exchange chromatography, and be adjusted to the ultimate density of 1.5M ammonium sulfate and 50mM phosphate buffer pH 7.0 (electrical conductivity 216mS/cm) by adding 2M ammonium sulfate and 50mM phosphate buffer.Then sample is added to hydrophobic interaction post (for example, HiTrap Phenyl HP, GE Healthcare), this post is the balance under the flow velocity of 60ml/h by 1.5M ammonium sulfate and 50mM phosphate buffer (pH 7.0) in advance.Behind the application of sample, (reduce to 0M from 1.5M, the material that 35CV) comes wash-out to keep by reduce ammonium sulfate concentrations with linear mode.Can determine the fB3 that exists in the flow point by for example SDS-PAGE, Western trace and/or ELISA.

The peak that will contain fB3 then is concentrated into the final volume of 1ml and it is carried out gel filtration respectively and balance in PBS buffer solution (pH 7.0 for 50mM phosphate, 150mMNaCl) on the SephacrylS30016/26HR post.Carry out the wash-out of fB3 and detect in 280nm monitoring stream fluid at the constant linear flow velocity of 30cm/h by UV.Can for example determine flow point purity by the SDS-PAGE that adopts silver dyeing.Can use the haemolysis test in the presence of wild type fB, to carry out activity analysis.

In addition, can be with the PD-10 desalting column with through the flow point of the fB3 of HIC purifying or merge flow point and exchange in the GVB buffer solution.Thereby can carry out haemolysis test detection by the fB3 that adds increase dosage and suppress active through the HIC purifying.

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Should be appreciated that, be conspicuous to the variations and modifications of embodiment as herein described for those skilled in the art.Can carry out these variations and modification and do not deviate from the spirit and scope of theme of the present invention and can not weaken its expection advantage.

By with reference to its full content is introduced in this specification, its degree is equal to specifically and indicates the publication that each is independent, patent and patent application individually by with reference to being incorporated herein at this in all publications, patent and the patent application of mentioning in this specification.

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＜110〉Advanced Vision Therapies Inc. (US) (ADVANCED VISION THERAPIES, INC.)

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<400>2

Met?Gly?Ser?Asn?Leu?Ser?Pro?Gln?Leu?Cys?Leu?Met?Pro?Phe?Ile?Leu

1???????????????5???????????????????10??????????????????15

Gly?Leu?Leu?Ser?Gly?Gly?Val?Thr?Thr?Thr?Pro?Trp?Ser?Leu?Ala?Arg

20??????????????????25??????????????????30

Pro?Gln?Gly?Ser?Cys?Ser?Leu?Glu?Gly?Val?Glu?Ile?Lys?Gly?Gly?Ser

35??????????????????40??????????????????45

Phe?Arg?Leu?Leu?Gln?Glu?Gly?Gln?Ala?Leu?Glu?Tyr?Val?Cys?Pro?Ser

50??????????????????55??????????????????60

Gly?Phe?Tyr?Pro?Tyr?Pro?Val?Gln?Thr?Arg?Thr?Cys?Arg?Ser?Thr?Gly

65??????????????????70??????????????????75??????????????????80

Ser?Trp?Ser?Thr?Leu?Lys?Thr?Gln?Asp?Gln?Lys?Thr?Val?Arg?Lys?Ala

85??????????????????90??????????????????95

Glu?Cys?Arg?Ala?Ile?His?Cys?Pro?Arg?Pro?His?Asp?Phe?Glu?Asn?Gly

100?????????????????105?????????????????110

Glu?Tyr?Trp?Pro?Arg?Ser?Pro?Tyr?Tyr?Asn?Val?Ser?Asp?Glu?Ile?Ser

115?????????????????120?????????????????125

Phe?His?Cys?Tyr?Asp?Gly?Tyr?Thr?Leu?Arg?Gly?Ser?Ala?Asn?Arg?Thr

130?????????????????135?????????????????140

Cys?Gln?Val?Asn?Gly?Arg?Trp?Ser?Gly?Gln?Thr?Ala?Ile?Cys?Asp?Asn

145?????????????????150?????????????????155?????????????????160

Gly?Ala?Gly?Tyr?Cys?Ser?Asn?Pro?Gly?Ile?Pro?Ile?Gly?Thr?Arg?Lys

165?????????????????170?????????????????175

Val?Gly?Ser?Gln?Tyr?Arg?Leu?Glu?Asp?Ser?Val?Thr?Tyr?His?Cys?Ser

180?????????????????185?????????????????190

Arg?Gly?Leu?Thr?Leu?Arg?Gly?Ser?Gln?Arg?Arg?Thr?Cys?Gln?Glu?Gly

195?????????????????200?????????????????205

Gly?Ser?Trp?Ser?Gly?Thr?Glu?Pro?Ser?Cys?Gln?Asp?Ser?Phe?Met?Tyr

210?????????????????215?????????????????220

Asp?Thr?Pro?Gln?Glu?Val?Ala?Glu?Ala?Phe?Leu?Ser?Ser?Leu?Thr?Glu

225?????????????????230?????????????????235?????????????????240

Thr?Ile?Glu?Gly?Val?Asp?Ala?Glu?Asp?Gly?His?Gly?Pro?Gly?Glu?Gln

245?????????????????250?????????????????255

Gln?Lys?Arg?Lys?Ile?Val?Leu?Asp?Pro?Ser?Gly?Ser?Met?Asn?Ile?Tyr

260?????????????????265?????????????????270

Leu?Val?Leu?Asp?Gly?Ser?Asp?Ser?Ile?Gly?Ala?Ser?Asn?Phe?Thr?Gly

275?????????????????280?????????????????285

Ala?Lys?Lys?Cys?Leu?Val?Asn?Leu?Ile?Glu?Lys?Val?Ala?Ser?Tyr?Gly

290?????????????????295?????????????????300

Val?Lys?Pro?Arg?Tyr?Gly?Leu?Val?Thr?Tyr?Ala?Thr?Tyr?Pro?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Trp?Val?Lys?Val?Ser?Glu?Ala?Asp?Ser?Ser?Asn?Ala?Asp?Trp?Val?Thr

325?????????????????330?????????????????335

Lys?Gln?Leu?Asn?Glu?Ile?Asn?Tyr?Glu?Asp?His?Lys?Leu?Lys?Ser?Gly

340?????????????????345?????????????????350

Thr?Asn?Thr?Lys?Lys?Ala?Leu?Gln?Ala?Val?Tyr?Ser?Met?Met?Ser?Trp

355?????????????????360?????????????????365

Pro?Asp?Asp?Val?Pro?Pro?Glu?Gly?Trp?Asn?Arg?Thr?Arg?His?Val?Ile

370?????????????????375?????????????????380

Ile?Leu?Met?Thr?Asp?Gly?Leu?His?Asn?Met?Gly?Gly?Asp?Pro?Ile?Thr

385?????????????????390?????????????????395?????????????????400

Val?Ile?Asp?Glu?Ile?Arg?Asp?Leu?Leu?Tyr?Ile?Gly?Lys?Asp?Arg?Lys

405?????????????????410?????????????????415

Asn?Pro?Arg?Glu?Asp?Tyr?Leu?Asp?Val?Tyr?Val?Phe?Gly?Val?Gly?Pro

420?????????????????425?????????????????430

Leu?Val?Asn?Gln?Val?Asn?Ile?Asn?Ala?Leu?Ala?Ser?Lys?Lys?Asp?Asn

435?????????????????440?????????????????445

Glu?Gln?His?Val?Phe?Lys?Val?Lys?Asp?Met?Glu?Asn?Leu?Glu?Asp?Val

450?????????????????455?????????????????460

Phe?Tyr?Gln?Met?Ile?Asp?Glu?Ser?Gln?Ser?Leu?Ser?Leu?Cys?Gly?Met

465?????????????????470?????????????????475?????????????????480

Val?Trp?Glu?His?Arg?Lys?Gly?Thr?Asp?Tyr?His?Lys?Gln?Pro?Trp?Gln

485?????????????????490?????????????????495

Ala?Lys?Ile?Ser?Val?Ile?Arg?Pro?Ser?Lys?Gly?His?Glu?Ser?Cys?Met

500?????????????????505?????????????????510

Gly?Ala?Val?Val?Ser?Glu?Tyr?Phe?Val?Leu?Thr?Ala?Ala?His?Cys?Phe

515?????????????????520?????????????????525

Thr?Val?Asp?Asp?Lys?Glu?His?Ser?Ile?Lys?Val?Ser?Val?Gly?Gly?Glu

530?????????????????535?????????????????540

Lys?Arg?Asp?Leu?Glu?Ile?Glu?Val?Val?Leu?Phe?His?Pro?Asn?Tyr?Asn

545?????????????????550?????????????????555?????????????????560

Ile?Asn?Gly?Lys?Lys?Glu?Ala?Gly?Ile?Pro?Glu?Phe?Tyr?Asp?Tyr?Asp

565?????????????????570?????????????????575

Val?Ala?Leu?Ile?Lys?Leu?Lys?Asn?Lys?Leu?Lys?Tyr?Gly?Gln?Thr?Ile

580?????????????????585?????????????????590

Arg?Pro?Ile?Cys?Leu?Pro?Cys?Thr?Glu?Gly?Thr?Thr?Arg?Ala?Leu?Arg

595?????????????????600?????????????????605

Leu?Pro?Pro?Thr?Thr?Thr?Cys?Gln?Gln?Gln?Lys?Glu?Glu?Leu?Leu?Pro

610?????????????????615?????????????????620

Ala?Gln?Asp?Ile?Lys?Ala?Leu?Phe?Val?Ser?Glu?Glu?Glu?Lys?Lys?Leu

625?????????????????630?????????????????635?????????????????640

Thr?Arg?Lys?Glu?Val?Tyr?Ile?Lys?Asn?Gly?Asp?Lys?Lys?Gly?Ser?Cys

645?????????????????650?????????????????655

Glu?Arg?Asp?Ala?Gln?Tyr?Ala?Pro?Gly?Tyr?Asp?Lys?Val?Lys?Asp?Ile

660?????????????????665?????????????????670

Ser?Glu?Val?Val?Thr?Pro?Arg?Phe?Leu?Cys?Thr?Gly?Gly?Val?Ser?Pro

675?????????????????680?????????????????685

Tyr?Ala?Asp?Pro?Asn?Thr?Cys?Arg?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ile

690?????????????????695?????????????????700

Val?His?Lys?Arg?Ser?Arg?Phe?Ile?Gln?Val?Gly?Val?Ile?Ser?Trp?Gly

705?????????????????710?????????????????715?????????????????720

Val?Val?Asp?Val?Cys?Lys?Asn?Gln?Lys?Arg?Gln?Lys?Gln?Val?Pro?Ala

725?????????????????730?????????????????735

His?Ala?Arg?Asp?Phe?His?Ile?Asn?Leu?Phe?Gln?Val?Leu?Pro?Trp?Leu

740?????????????????745?????????????????750

Lys?Glu?Lys?Leu?Gln?Asp?Glu?Asp?Leu?Gly?Phe?Leu

755?????????????????760

<210>3

<211>2368

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>3

tcctgcccca?ggcccagctt?ctctcctgcc?ttccaacgcc?atggggagca?atctcagccc????60

ccaactctgc?ctgatgccct?ttatcttggg?cctcttgtct?ggaggtgtga?ccaccactcc???120

atggtctttg?gcccggcccc?agggatcctg?ctctctggag?ggggtagaga?tcaaaggcgg???180

ctccttccga?cttctccaag?agggccaggc?actggagtac?gtgtgtcctt?ctggcttcta???240

cccgtaccct?gtgcagacac?gtacctgcag?atctacgggg?tcctggagca?ccctgaagac???300

tcaagaccaa?aagactgtca?ggaaggcaga?gtgcagagca?atccactgtc?caagaccaca???360

cgacttcgag?aacggggaat?actggccccg?gtctccctac?tacaatgtga?gtgatgagat???420

ctctttccac?tgctatgacg?gttacactct?ccggggctct?gccaatcgca?cctgccaagt???480

gaatggccgg?tggagtgggc?agacagcgat?ctgtgacaac?ggagcggggt?actgctccaa???540

cccgggcatc?cccattggca?caaggaaggt?gggcagccag?taccgccttg?aagacagcgt???600

cacctaccac?tgcagccggg?ggcttaccct?gcgtggctcc?cagcggcgaa?cgtgtcagga???660

aggtggctct?tggagcggga?cggagccttc?ctgccaagac?tccttcatgt?acgacacccc???720

tcaagaggtg?gccgaagctt?tcctgtcttc?cctgacagag?accatagaag?gagtcgatgc???780

tgaggatggg?cacggcccag?gggaacaaca?gaagcggaag?atcgtcctgg?acccttcagg???840

ctccatgaac?atctacctgg?tgctagatgg?atcagacagc?attggggcca?gcaacttcac???900

aggagccaaa?aagtgtctag?tcaacttaat?tgagaaggtg?gcaagttatg?gtgtgaagcc???960

aagatatggt?ctagtgacat?atgccacata?ccccaaaatt?tgggtcaaag?tgtctgaagc??1020

agacagcagt?aatgcagact?gggtcacgaa?gcagctcaat?gaaatcaatt?atgaagacca??1080

caagttgaag?tcagggacta?acaccaagaa?ggccctccag?gcagtgtaca?gcatgatgag??1140

ctggccagat?gacgtccctc?ctgaaggctg?gaaccgcacc?cgccatgtca?tcatcctcat??1200

gactgatgga?ttgcacaaca?tgggcgggga?cccaattact?gtcattgatg?agatccggga??1260

cttgctatac?attggcaagg?atcgcaaaaa?cccaagggag?gattatctgg?atgtctatgt??1320

gtttggggtc?gggcctttgg?tgaaccaagt?gaacatcaat?gctttggctt?ccaagaaaga??1380

caatgagcaa?catgtgttca?aagtcaagga?tatggaaaac?ctggaagatg?ttttctacca??1440

aatgatcgat?gaaagccagt?ctctgagtct?ctgtggcatg?gtttgggaac?acaggaaggg??1500

taccgattac?cacaagcaac?catggcaggc?caagatctca?gtcattcgcc?cttcaaaggg??1560

acacgagagc?tgtatggggg?ctgtggtgtc?tgagtacttt?gtgctgacag?cagcacattg??1620

tttcactgtg?gatgacaagg?aacactcaat?caaggtcagc?gtaggagggg?agaagcggga??1680

cctggagata?gaagtagtcc?tatttcaccc?caactacaac?attaatggga?aaaaagaagc??1740

aggaattcct?gaattttatg?actatgacgt?tgccctgatc?aagctcaaga?ataagctgaa??1800

atatggccag?actatcaggc?ccatttgtct?cccctgcacc?gagggaacaa?ctcgagcttt??1860

gaggcttcct?ccaactacca?cttgccagca?acaaaaggaa?gagctgctcc?ctgcacagga??1920

tatcaaagct?ctgtttgtgt?ctgaggagga?gaaaaagctg?actcggaagg?aggtctacat??1980

caagaatggg?gataagaaag?gcagctgtga?gagagatgct?caatatgccc?caggctatga??2040

caaagtcaag?gacatctcag?aggtggtcac?ccctcggttc?ctttgtactg?gaggagtgag??2100

tccctatgct?gaccccaata?cttgcagagg?tgattctggc?ggccccttga?tagttcacaa??2160

gagaagtcgt?ttcattcaag?ttggtgtaat?cagctgggga?gtagtggatg?tctgcaaaaa??2220

ccagaagcgg?caaaagcagg?tacctgctca?cgcccgaaac?tttcacatca?acctctttca??2280

agtgctgccc?tggctgaagg?agaaactcca?agatgaggat?ttgggttttc?tatgaggggt??2340

ttcctgctgg?acaggggcgt?gggattga?????????????????????????????????????2368

<210>4

<211>764

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>4

Met?Gly?Ser?Asn?Leu?Ser?Pro?Gln?Leu?Cys?Leu?Met?Pro?Phe?Ile?Leu

1???????????????5???????????????????10??????????????????15

Gly?Leu?Leu?Ser?Gly?Gly?Val?Thr?Thr?Thr?Pro?Trp?Ser?Leu?Ala?Arg

20??????????????????25??????????????????30

Pro?Gln?Gly?Ser?Cys?Ser?Leu?Glu?Gly?Val?Glu?Ile?Lys?Gly?Gly?Ser

35??????????????????40??????????????????45

Phe?Arg?Leu?Leu?Gln?Glu?Gly?Gln?Ala?Leu?Glu?Tyr?Val?Cys?Pro?Ser

50??????????????????55??????????????????60

Gly?Phe?Tyr?Pro?Tyr?Pro?Val?Gln?Thr?Arg?Thr?Cys?Arg?Ser?Thr?Gly

65??????????????????70??????????????????75??????????????????80

Ser?Trp?Ser?Thr?Leu?Lys?Thr?Gln?Asp?Gln?Lys?Thr?Val?Arg?Lys?Ala

85??????????????????90??????????????????95

Glu?Cys?Arg?Ala?Ile?His?Cys?Pro?Arg?Pro?His?Asp?Phe?Glu?Asn?Gly

100?????????????????105?????????????????110

Glu?Tyr?Trp?Pro?Arg?Ser?Pro?Tyr?Tyr?Asn?Val?Ser?Asp?Glu?Ile?Ser

115?????????????????120?????????????????125

Phe?His?Cys?Tyr?Asp?Gly?Tyr?Thr?Leu?Arg?Gly?Ser?Ala?Asn?Arg?Thr

130?????????????????135?????????????????140

Cys?Gln?Val?Asn?Gly?Arg?Trp?Ser?Gly?Gln?Thr?Ala?Ile?Cys?Asp?Asn

145?????????????????150?????????????????155?????????????????160

Gly?Ala?Gly?Tyr?Cys?Ser?Asn?Pro?Gly?Ile?Pro?Ile?Gly?Thr?Arg?Lys

165?????????????????170?????????????????175

Val?Gly?Ser?Gln?Tyr?Arg?Leu?Glu?Asp?Ser?Val?Thr?Tyr?His?Cys?Ser

180?????????????????185?????????????????190

Arg?Gly?Leu?Thr?Leu?Arg?Gly?Ser?Gln?Arg?Arg?Thr?Cys?Gln?Glu?Gly

195?????????????????200?????????????????205

Gly?Ser?Trp?Ser?Gly?Thr?Glu?Pro?Ser?Cys?Gln?Asp?Ser?Phe?Met?Tyr

210?????????????????215?????????????????220

Asp?Thr?Pro?Gln?Glu?Val?Ala?Glu?Ala?Phe?Leu?Ser?Ser?Leu?Thr?Glu

225?????????????????230?????????????????235?????????????????240

Thr?Ile?Glu?Gly?Val?Asp?Ala?Glu?Asp?Gly?His?Gly?Pro?Gly?Glu?Gln

245?????????????????250?????????????????255

Gln?Lys?Arg?Lys?Ile?Val?Leu?Asp?Pro?Ser?Gly?Ser?Met?Asn?Ile?Tyr

260?????????????????265?????????????????270

Leu?Val?Leu?Asp?Gly?Ser?Asp?Ser?Ile?Gly?Ala?Ser?Asn?Phe?Thr?Gly

275?????????????????280?????????????????285

Ala?Lys?Lys?Cys?Leu?Val?Asn?Leu?Ile?Glu?Lys?Val?Ala?Ser?Tyr?Gly

290?????????????????295?????????????????300

Val?Lys?Pro?Arg?Tyr?Gly?Leu?Val?Thr?Tyr?Ala?Thr?Tyr?Pro?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Trp?Val?Lys?Val?Ser?Glu?Ala?Asp?Ser?Ser?Asn?Ala?Asp?Trp?Val?Thr

325?????????????????330?????????????????335

Lys?Gln?Leu?Asn?Glu?Ile?Asn?Tyr?Glu?Asp?His?Lys?Leu?Lys?Ser?Gly

340?????????????????345?????????????????350

Thr?Asn?Thr?Lys?Lys?Ala?Leu?Gln?Ala?Val?Tyr?Ser?Met?Met?Ser?Trp

355?????????????????360?????????????????365

Pro?Asp?Asp?Val?Pro?Pro?Glu?Gly?Trp?Asn?Arg?Thr?Arg?His?Val?Ile

370?????????????????375?????????????????380

Ile?Leu?Met?Thr?Asp?Gly?Leu?His?Asn?Met?Gly?Gly?Asp?Pro?Ile?Thr

385?????????????????390?????????????????395?????????????????400

Val?Ile?Asp?Glu?Ile?Arg?Asp?Leu?Leu?Tyr?Ile?Gly?Lys?Asp?Arg?Lys

405?????????????????410?????????????????415

Asn?Pro?Arg?Glu?Asp?Tyr?Leu?Asp?Val?Tyr?Val?Phe?Gly?Val?Gly?Pro

420?????????????????425?????????????????430

Leu?Val?Asn?Gln?Val?Asn?Ile?Asn?Ala?Leu?Ala?Ser?Lys?Lys?Asp?Asn

435?????????????????440?????????????????445

Glu?Gln?His?Val?Phe?Lys?Val?Lys?Asp?Met?Glu?Asn?Leu?Glu?Asp?Val

450?????????????????455?????????????????460

Phe?Tyr?Gln?Met?Ile?Asp?Glu?Ser?Gln?Ser?Leu?Ser?Leu?Cys?Gly?Met

465?????????????????470?????????????????475?????????????????480

Val?Trp?Glu?His?Arg?Lys?Gly?Thr?Asp?Tyr?His?Lys?Gln?Pro?Trp?Gln

485?????????????????490?????????????????495

Ala?Lys?Ile?Ser?Val?Ile?Arg?Pro?Ser?Lys?Gly?His?Glu?Ser?Cys?Met

500?????????????????505?????????????????510

Gly?Ala?Val?Val?Ser?Glu?Tyr?Phe?Val?Leu?Thr?Ala?Ala?His?Cys?Phe

515?????????????????520?????????????????525

Thr?Val?Asp?Asp?Lys?Glu?His?Ser?Ile?Lys?Val?Ser?Val?Gly?Gly?Glu

530?????????????????535?????????????????540

Lys?Arg?Asp?Leu?Glu?Ile?Glu?Val?Val?Leu?Phe?His?Pro?Asn?Tyr?Asn

545?????????????????550?????????????????555?????????????????560

Ile?Asn?Gly?Lys?Lys?Glu?Ala?Gly?Ile?Pro?Glu?Phe?Tyr?Asp?Tyr?Asp

565?????????????????570?????????????????575

Val?Ala?Leu?Ile?Lys?Leu?Lys?Asn?Lys?Leu?Lys?Tyr?Gly?Gln?Thr?Ile

580?????????????????585?????????????????590

Arg?Pro?Ile?Cys?Leu?Pro?Cys?Thr?Glu?Gly?Thr?Thr?Arg?Ala?Leu?Arg

595?????????????????600?????????????????605

Leu?Pro?Pro?Thr?Thr?Thr?Cys?Gln?Gln?Gln?Lys?Glu?Glu?Leu?Leu?Pro

610?????????????????615?????????????????620

Ala?Gln?Asp?Ile?Lys?Ala?Leu?Phe?Val?Ser?Glu?Glu?Glu?Lys?Lys?Leu

625?????????????????630?????????????????635?????????????????640

Thr?Arg?Lys?Glu?Val?Tyr?Ile?Lys?Asn?Gly?Asp?Lys?Lys?Gly?Ser?Cys

645?????????????????650?????????????????655

Glu?Arg?Asp?Ala?Gln?Tyr?Ala?Pro?Gly?Tyr?Asp?Lys?Val?Lys?Asp?Ile

660?????????????????665?????????????????670

Ser?Glu?Val?Val?Thr?Pro?Arg?Phe?Leu?Cys?Thr?Gly?Gly?Val?Ser?Pro

675?????????????????680?????????????????685

Tyr?Ala?Asp?Pro?Asn?Thr?Cys?Arg?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ile

690?????????????????695?????????????????700

Val?His?Lys?Arg?Ser?Arg?Phe?Ile?Gln?Val?Gly?Val?Ile?Ser?Trp?Gly

705?????????????????710?????????????????715?????????????????720

Val?Val?Asp?Val?Cys?Lys?Asn?Gln?Lys?Arg?Gln?Lys?Gln?Val?Pro?Ala

725?????????????????730?????????????????735

His?Ala?Arg?Asn?Phe?His?Ile?Asn?Leu?Phe?Gln?Val?Leu?Pro?Trp?Leu

740?????????????????745?????????????????750

Lys?Glu?Lys?Leu?Gln?Asp?Glu?Asp?Leu?Gly?Phe?Leu

755?????????????????760

<210>5

<211>2368

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>5

tcctgcccca?ggcccagctt?ctctcctgcc?ttccaacgcc?atggggagca?atctcagccc????60

ccaactctgc?ctgatgccct?ttatcttggg?cctcttgtct?ggaggtgtga?ccaccactcc???120

atggtctttg?gcccggcccc?agggatcctg?ctctctggag?ggggtagaga?tcaaaggcgg???180

ctccttccga?cttctccaag?agggccaggc?actggagtac?gtgtgtcctt?ctggcttcta???240

cccgtaccct?gtgcagacac?gtacctgcag?atctacgggg?tcctggagca?ccctgaagac???300

tcaagaccaa?aagactgtca?ggaaggcaga?gtgcagagca?atccactgtc?caagaccaca???360

cgacttcgag?aacggggaat?actggccccg?gtctccctac?tacaatgtga?gtgatgagat???420

ctctttccac?tgctatgacg?gttacactct?ccggggctct?gccaatcgca?cctgccaagt???480

gaatggccgg?tggagtgggc?agacagcgat?ctgtgacaac?ggagcggggt?actgctccaa???540

cccgggcatc?cccattggca?caaggaaggt?gggcagccag?taccgccttg?aagacagcgt???600

cacctaccac?tgcagccggg?ggcttaccct?gcgtggctcc?cagcggcgaa?cgtgtcagga???660

aggtggctct?tggagcggga?cggagccttc?ctgccaagac?tccttcatgt?acgacacccc???720

tcaagaggtg?gccgaagctt?tcctgtcttc?cctgacagag?accatagaag?gagtcgatgc???780

tgaggatggg?cacggcccag?gggaacaaca?gaagcggaag?atcgtcctgg?acccttcagg???840

ctccatgaac?atctacctgg?tgctagatgg?atcaggcagc?attggggcca?gcgacttcac???900

aggagccaaa?aagtgtctag?tcaacttaat?tgagaaggtg?gcaagttatg?gtgtgaagcc???960

aagatatggt?ctagtgacat?atgccacata?ccccaaaatt?tgggtcaaag?tgtctgaagc??1020

agacagcagt?aatgcagact?gggtcacgaa?gcagctcaat?gaaatcaatt?atgaagacca??1080

caagttgaag?tcagggacta?acaccaagaa?ggccctccag?gcagtgtaca?gcatgatgag??1140

ctggccagat?gacgtccctc?ctgaaggctg?gaaccgcacc?cgccatgtca?tcatcctcat??1200

gactgatgga?ttgcacaaca?tgggcgggga?cccaattact?gtcattgatg?agatccggga??1260

cttgctatac?attggcaagg?atcgcaaaaa?cccaagggag?gattatctgg?atgtctatgt??1320

gtttggggtc?gggcctttgg?tgaaccaagt?gaacatcaat?gctttggctt?ccaagaaaga??1380

caatgagcaa?catgtgttca?aagtcaagga?tatggaaaac?ctggaagatg?ttttctacca??1440

aatgatcgat?gaaagccagt?ctctgagtct?ctgtggcatg?gtttgggaac?acaggaaggg??1500

taccgattac?cacaagcaac?catggcaggc?caagatctca?gtcattcgcc?cttcaaaggg??1560

acacgagagc?tgtatggggg?ctgtggtgtc?tgagtacttt?gtgctgacag?cagcacattg??1620

tttcactgtg?gatgacaagg?aacactcaat?caaggtcagc?gtaggagggg?agaagcggga??1680

cctggagata?gaagtagtcc?tatttcaccc?caactacaac?attaatggga?aaaaagaagc??1740

aggaattcct?gaattttatg?actatgacgt?tgccctgatc?aagctcaaga?ataagctgaa??1800

atatggccag?actatcaggc?ccatttgtct?cccctgcacc?gagggaacaa?ctcgagcttt??1860

gaggcttcct?ccaactacca?cttgccagca?acaaaaggaa?gagctgctcc?ctgcacagga??1920

tatcaaagct?ctgtttgtgt?ctgaggagga?gaaaaagctg?actcggaagg?aggtctacat??1980

caagaatggg?gataagaaag?gcagctgtga?gagagatgct?caatatgccc?caggctatga??2040

caaagtcaag?gacatctcag?aggtggtcac?ccctcggttc?ctttgtactg?gaggagtgag??2100

tccctatgct?gaccccaata?cttgcagagg?tgattctggc?ggccccttga?tagttcacaa??2160

gagaagtcgt?ttcattcaag?ttggtgtaat?cagctgggga?gtagtggatg?tctgcaaaaa??2220

ccagaagcgg?caaaagcagg?tacctgctca?cgcccgaaac?tttcacatca?acctctttca??2280

agtgctgccc?tggctgaagg?agaaactcca?agatgaggat?ttgggttttc?tatgaggggt??2340

ttcctgctgg?acaggggcgt?gggattga?????????????????????????????????????2368

<210>6

<211>764

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>6

Met?Gly?Ser?Asn?Leu?Ser?Pro?Gln?Leu?Cys?Leu?Met?Pro?Phe?Ile?Leu

1???????????????5???????????????????10??????????????????15

Gly?Leu?Leu?Ser?Gly?Gly?Val?Thr?Thr?Thr?Pro?Trp?Ser?Leu?Ala?Arg

20??????????????????25??????????????????30

Pro?Gln?Gly?Ser?Cys?Ser?Leu?Glu?Gly?Val?Glu?Ile?Lys?Gly?Gly?Ser

35??????????????????40??????????????????45

Phe?Arg?Leu?Leu?Gln?Glu?Gly?Gln?Ala?Leu?Glu?Tyr?Val?Cys?Pro?Ser

50??????????????????55??????????????????60

Gly?Phe?Tyr?Pro?Tyr?Pro?Val?Gln?Thr?Arg?Thr?Cys?Arg?Ser?Thr?Gly

65??????????????????70??????????????????75??????????????????80

Ser?Trp?Ser?Thr?Leu?Lys?Thr?Gln?Asp?Gln?Lys?Thr?Val?Arg?Lys?Ala

85??????????????????90??????????????????95

Glu?Cys?Arg?Ala?Ile?His?Cys?Pro?Arg?Pro?His?Asp?Phe?Glu?Asn?Gly

100?????????????????105?????????????????110

Glu?Tyr?Trp?Pro?Arg?Ser?Pro?Tyr?Tyr?Asn?Val?Ser?Asp?Glu?Ile?Ser

115?????????????????120?????????????????125

Phe?His?Cys?Tyr?Asp?Gly?Tyr?Thr?Leu?Arg?Gly?Ser?Ala?Asn?Arg?Thr

130?????????????????135?????????????????140

Cys?Gln?Val?Asn?Gly?Arg?Trp?Ser?Gly?Gln?Thr?Ala?Ile?Cys?Asp?Asn

145?????????????????150?????????????????155?????????????????160

Gly?Ala?Gly?Tyr?Cys?Ser?Asn?Pro?Gly?Ile?Pro?Ile?Gly?Thr?Arg?Lys

165?????????????????170?????????????????175

Val?Gly?Ser?Gln?Tyr?Arg?Leu?Glu?Asp?Ser?Val?Thr?Tyr?His?Cys?Ser

180?????????????????185?????????????????190

Arg?Gly?Leu?Thr?Leu?Arg?Gly?Ser?Gln?Arg?Arg?Thr?Cys?Gln?Glu?Gly

195?????????????????200?????????????????205

Gly?Ser?Trp?Ser?Gly?Thr?Glu?Pro?Ser?Cys?Gln?Asp?Ser?Phe?Met?Tyr

210?????????????????215?????????????????220

Asp?Thr?Pro?Gln?Glu?Val?Ala?Glu?Ala?Phe?Leu?Ser?Ser?Leu?Thr?Glu

225?????????????????230?????????????????235?????????????????240

Thr?Ile?Glu?Gly?Val?Asp?Ala?Glu?Asp?Gly?His?Gly?Pro?Gly?Glu?Gln

245?????????????????250?????????????????255

Gln?Lys?Arg?Lys?Ile?Val?Leu?Asp?Pro?Ser?Gly?Ser?Met?Asn?Ile?Tyr

260?????????????????265?????????????????270

Leu?Val?Leu?Asp?Gly?Ser?Gly?Ser?Ile?Gly?Ala?Ser?Asp?Phe?Thr?Gly

275?????????????????280?????????????????285

Ala?Lys?Lys?Cys?Leu?Val?Asn?Leu?Ile?Glu?Lys?Val?Ala?Ser?Tyr?Gly

290?????????????????295?????????????????300

Val?Lys?Pro?Arg?Tyr?Gly?Leu?Val?Thr?Tyr?Ala?Thr?Tyr?Pro?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Trp?Val?Lys?Val?Ser?Glu?Ala?Asp?Ser?Ser?Asn?Ala?Asp?Trp?Val?Thr

325?????????????????330?????????????????335

Lys?Gln?Leu?Asn?Glu?Ile?Asn?Tyr?Glu?Asp?His?Lys?Leu?Lys?Ser?Gly

340?????????????????345?????????????????350

Thr?Asn?Thr?Lys?Lys?Ala?Leu?Gln?Ala?Val?Tyr?Ser?Met?Met?Ser?Trp

355?????????????????360?????????????????365

Pro?Asp?Asp?Val?Pro?Pro?Glu?Gly?Trp?Asn?Arg?Thr?Arg?His?Val?Ile

370?????????????????375?????????????????380

Ile?Leu?Met?Thr?Asp?Gly?Leu?His?Asn?Met?Gly?Gly?Asp?Pro?Ile?Thr

385?????????????????390?????????????????395?????????????????400

Val?Ile?Asp?Glu?Ile?Arg?Asp?Leu?Leu?Tyr?Ile?Gly?Lys?Asp?Arg?Lys

405?????????????????410?????????????????415

Asn?Pro?Arg?Glu?Asp?Tyr?Leu?Asp?Val?Tyr?Val?Phe?Gly?Val?Gly?Pro

420?????????????????425?????????????????430

Leu?Val?Asn?Gln?Val?Asn?Ile?Asn?Ala?Leu?Ala?Ser?Lys?Lys?Asp?Asn

435?????????????????440?????????????????445

Glu?Gln?His?Val?Phe?Lys?Val?Lys?Asp?Met?Glu?Asn?Leu?Glu?Asp?Val

450?????????????????455?????????????????460

Phe?Tyr?Gln?Met?Ile?Asp?Glu?Ser?Gln?Ser?Leu?Ser?Leu?Cys?Gly?Met

465?????????????????470?????????????????475?????????????????480

Val?Trp?Glu?His?Arg?Lys?Gly?Thr?Asp?Tyr?His?Lys?Gln?Pro?Trp?Gln

485?????????????????490?????????????????495

Ala?Lys?Ile?Ser?Val?Ile?Arg?Pro?Ser?Lys?Gly?His?Glu?Ser?Cys?Met

500?????????????????505?????????????????510

Gly?Ala?Val?Val?Ser?Glu?Tyr?Phe?Val?Leu?Thr?Ala?Ala?His?Cys?Phe

515?????????????????520?????????????????525

Thr?Val?Asp?Asp?Lys?Glu?His?Ser?Ile?Lys?Val?Ser?Val?Gly?Gly?Glu

530?????????????????535?????????????????540

Lys?Arg?Asp?Leu?Glu?Ile?Glu?Val?Val?Leu?Phe?His?Pro?Asn?Tyr?Asn

545?????????????????550?????????????????555?????????????????560

Ile?Asn?Gly?Lys?Lys?Glu?Ala?Gly?Ile?Pro?Glu?Phe?Tyr?Asp?Tyr?Asp

565?????????????????570?????????????????575

Val?Ala?Leu?Ile?Lys?Leu?Lys?Asn?Lys?Leu?Lys?Tyr?Gly?Gln?Thr?Ile

580?????????????????585?????????????????590

Arg?Pro?Ile?Cys?Leu?Pro?Cys?Thr?Glu?Gly?Thr?Thr?Arg?Ala?Leu?Arg

595?????????????????600?????????????????605

Leu?Pro?Pro?Thr?Thr?Thr?Cys?Gln?Gln?Gln?Lys?Glu?Glu?Leu?Leu?Pro

610?????????????????615?????????????????620

Ala?Gln?Asp?Ile?Lys?Ala?Leu?Phe?Val?Ser?Glu?Glu?Glu?Lys?Lys?Leu

625?????????????????630?????????????????635?????????????????640

Thr?Arg?Lys?Glu?Val?Tyr?Ile?Lys?Asn?Gly?Asp?Lys?Lys?Gly?Ser?Cys

645?????????????????650?????????????????655

Glu?Arg?Asp?Ala?Gln?Tyr?Ala?Pro?Gly?Tyr?Asp?Lys?Val?Lys?Asp?Ile

660?????????????????665?????????????????670

Ser?Glu?Val?Val?Thr?Pro?Arg?Phe?Leu?Cys?Thr?Gly?Gly?Val?Ser?Pro

675?????????????????680?????????????????685

Tyr?Ala?Asp?Pro?Asn?Thr?Cys?Arg?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ile

690?????????????????695?????????????????700

Val?His?Lys?Arg?Ser?Arg?Phe?Ile?Gln?Val?Gly?Val?Ile?Ser?Trp?Gly

705?????????????????710?????????????????715?????????????????720

Val?Val?Asp?Val?Cys?Lys?Asn?Gln?Lys?Arg?Gln?Lys?Gln?Val?Pro?Ala

725?????????????????730?????????????????735

His?Ala?Arg?Asn?Phe?His?Ile?Asn?Leu?Phe?Gln?Val?Leu?Pro?Trp?Leu

740?????????????????745?????????????????750

Lys?Glu?Lys?Leu?Gln?Asp?Glu?Asp?Leu?Gly?Phe?Leu

755?????????????????760

<210>7

<211>2368

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>7

tcctgcccca?ggcccagctt?ctctcctgcc?ttccaacgcc?atggggagca?atctcagccc????60

ccaactctgc?ctgatgccct?ttatcttggg?cctcttgtct?ggaggtgtga?ccaccactcc???120

atggtctttg?gcccggcccc?agggatcctg?ctctctggag?ggggtagaga?tcaaaggcgg???180

ctccttccga?cttctccaag?agggccaggc?actggagtac?gtgtgtcctt?ctggcttcta???240

cccgtaccct?gtgcagacac?gtacctgcag?atctacgggg?tcctggagca?ccctgaagac???300

tcaagaccaa?aagactgtca?ggaaggcaga?gtgcagagca?atccactgtc?caagaccaca???360

cgacttcgag?aacggggaat?actggccccg?gtctccctac?tacaatgtga?gtgatgagat???420

ctctttccac?tgctatgacg?gttacactct?ccggggctct?gccaatcgca?cctgccaagt???480

gaatggccgg?tggagtgggc?agacagcgat?ctgtgacaac?ggagcggggt?actgctccaa???540

cccgggcatc?cccattggca?caaggaaggt?gggcagccag?taccgccttg?aagacagcgt???600

cacctaccac?tgcagccggg?ggcttaccct?gcgtggctcc?cagcggcgaa?cgtgtcagga???660

aggtggctct?tggagcggga?cggagccttc?ctgccaagac?tccttcatgt?acgacacccc???720

tcaagaggtg?gccgaagctt?tcctgtcttc?cctgacagag?accatagaag?gagtcgatgc???780

tgaggatggg?cacggcccag?gggaacaaca?ggcggcagcg?atcgtcctgg?acccttcagg???840

ctccatgaac?atctacctgg?tgctagatgg?atcaggcagc?attggggcca?gcgacttcac???900

aggagccaaa?aagtgtctag?tcaacttaat?tgagaaggtg?gcaagttatg?gtgtgaagcc???960

aagatatggt?ctagtgacat?atgccacata?ccccaaaatt?tgggtcaaag?tgtctgaagc??1020

agacagcagt?aatgcagact?gggtcacgaa?gcagctcaat?gaaatcaatt?atgaagacca??1080

caagttgaag?tcagggacta?acaccaagaa?ggccctccag?gcagtgtaca?gcatgatgag??1140

ctggccagat?gacgtccctc?ctgaaggctg?gaaccgcacc?cgccatgtca?tcatcctcat??1200

gactgatgga?ttgcacaaca?tgggcgggga?cccaattact?gtcattgatg?agatccggga??1260

cttgctatac?attggcaagg?atcgcaaaaa?cccaagggag?gattatctgg?atgtctatgt??1320

gtttggggtc?gggcctttgg?tgaaccaagt?gaacatcaat?gctttggctt?ccaagaaaga??1380

caatgagcaa?catgtgttca?aagtcaagga?tatggaaaac?ctggaagatg?ttttctacca??1440

aatgatcgat?gaaagccagt?ctctgagtct?ctgtggcatg?gtttgggaac?acaggaaggg??1500

taccgattac?cacaagcaac?catggcaggc?caagatctca?gtcattcgcc?cttcaaaggg??1560

acacgagagc?tgtatggggg?ctgtggtgtc?tgagtacttt?gtgctgacag?cagcacattg??1620

tttcactgtg?gatgacaagg?aacactcaat?caaggtcagc?gtaggagggg?agaagcggga??1680

cctggagata?gaagtagtcc?tatttcaccc?caactacaac?attaatggga?aaaaagaagc??1740

aggaattcct?gaattttatg?actatgacgt?tgccctgatc?aagctcaaga?ataagctgaa??1800

atatggccag?actatcaggc?ccatttgtct?cccctgcacc?gagggaacaa?ctcgagcttt??1860

gaggcttcct?ccaactacca?cttgccagca?acaaaaggaa?gagctgctcc?ctgcacagga??1920

tatcaaagct?ctgtttgtgt?ctgaggagga?gaaaaagctg?actcggaagg?aggtctacat??1980

caagaatggg?gataagaaag?gcagctgtga?gagagatgct?caatatgccc?caggctatga??2040

caaagtcaag?gacatctcag?aggtggtcac?ccctcggttc?ctttgtactg?gaggagtgag??2100

tccctatgct?gaccccaata?cttgcagagg?tgattctggc?ggccccttga?tagttcacaa??2160

gagaagtcgt?ttcattcaag?ttggtgtaat?cagctgggga?gtagtggatg?tctgcaaaaa??2220

ccagaagcgg?caaaagcagg?tacctgctca?cgcccgagac?tttcacatca?acctctttca??2280

agtgctgccc?tggctgaagg?agaaactcca?agatgaggat?ttgggttttc?tatgaggggt??2340

ttcctgctgg?acaggggcgt?gggattga?????????????????????????????????????2368

<210>8

<211>764

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>8

Met?Gly?Ser?Asn?Leu?Ser?Pro?Gln?Leu?Cys?Leu?Met?Pro?Phe?Ile?Leu

1???????????????5???????????????????10??????????????????15

Gly?Leu?Leu?Ser?Gly?Gly?Val?Thr?Thr?Thr?Pro?Trp?Ser?Leu?Ala?Arg

20??????????????????25??????????????????30

Pro?Gln?Gly?Ser?Cys?Ser?Leu?Glu?Gly?Val?Glu?Ile?Lys?Gly?Gly?Ser

35??????????????????40??????????????????45

Phe?Arg?Leu?Leu?Gln?Glu?Gly?Gln?Ala?Leu?Glu?Tyr?Val?Cys?Pro?Ser

50??????????????????55??????????????????60

Gly?Phe?Tyr?Pro?Tyr?Pro?Val?Gln?Thr?Arg?Thr?Cys?Arg?Ser?Thr?Gly

65??????????????????70??????????????????75??????????????????80

Ser?Trp?Ser?Thr?Leu?Lys?Thr?Gln?Asp?Gln?Lys?Thr?Val?Arg?Lys?Ala

85??????????????????90??????????????????95

Glu?Cys?Arg?Ala?Ile?His?Cys?Pro?Arg?Pro?His?Asp?Phe?Glu?Asn?Gly

100?????????????????105?????????????????110

Glu?Tyr?Trp?Pro?Arg?Ser?Pro?Tyr?Tyr?Asn?Val?Ser?Asp?Glu?Ile?Ser

115?????????????????120?????????????????125

Phe?His?Cys?Tyr?Asp?Gly?Tyr?Thr?Leu?Arg?Gly?Ser?Ala?Asn?Arg?Thr

130?????????????????135?????????????????140

Cys?Gln?Val?Asn?Gly?Arg?Trp?Ser?Gly?Gln?Thr?Ala?Ile?Cys?Asp?Asn

145?????????????????150?????????????????155?????????????????160

Gly?Ala?Gly?Tyr?Cys?Ser?Asn?Pro?Gly?Ile?Pro?Ile?Gly?Thr?Arg?Lys

165?????????????????170?????????????????175

Val?Gly?Ser?Gln?Tyr?Arg?Leu?Glu?Asp?Ser?Val?Thr?Tyr?His?Cys?Ser

180?????????????????185?????????????????190

Arg?Gly?Leu?Thr?Leu?Arg?Gly?Ser?Gln?Arg?Arg?Thr?Cys?Gln?Glu?Gly

195?????????????????200?????????????????205

Gly?Ser?Trp?Ser?Gly?Thr?Glu?Pro?Ser?Cys?Gln?Asp?Ser?Phe?Met?Tyr

210?????????????????215?????????????????220

Asp?Thr?Pro?Gln?Glu?Val?Ala?Glu?Ala?Phe?Leu?Ser?Ser?Leu?Thr?Glu

225?????????????????230?????????????????235?????????????????240

Thr?Ile?Glu?Gly?Val?Asp?Ala?Glu?Asp?Gly?His?Gly?Pro?Gly?Glu?Gln

245?????????????????250?????????????????255

Gln?Ala?Ala?Ala?Ile?Val?Leu?Asp?Pro?Ser?Gly?Ser?Met?Asn?Ile?Tyr

260?????????????????265?????????????????270

Leu?Val?Leu?Asp?Gly?Ser?Gly?Ser?Ile?Gly?Ala?Ser?Asp?Phe?Thr?Gly

275?????????????????280?????????????????285

Ala?Lys?Lys?Cys?Leu?Val?Asn?Leu?Ile?Glu?Lys?Val?Ala?Ser?Tyr?Gly

290?????????????????295?????????????????300

Val?Lys?Pro?Arg?Tyr?Gly?Leu?Val?Thr?Tyr?Ala?Thr?Tyr?Pro?Lys?Ile

305?????????????????310?????????????????315?????????????????320

Trp?Val?Lys?Val?Ser?Glu?Ala?Asp?Ser?Ser?Asn?Ala?Asp?Trp?Val?Thr

325?????????????????330?????????????????335

Lys?Gln?Leu?Asn?Glu?Ile?Asn?Tyr?Glu?Asp?His?Lys?Leu?Lys?Ser?Gly

340?????????????????345?????????????????350

Thr?Asn?Thr?Lys?Lys?Ala?Leu?Gln?Ala?Val?Tyr?Ser?Met?Met?Ser?Trp

355?????????????????360?????????????????365

Pro?Asp?Asp?Val?Pro?Pro?Glu?Gly?Trp?Asn?Arg?Thr?Arg?His?Val?Ile

370?????????????????375?????????????????380

Ile?Leu?Met?Thr?Asp?Gly?Leu?His?Asn?Met?Gly?Gly?Asp?Pro?Ile?Thr

385?????????????????390?????????????????395?????????????????400

Val?Ile?Asp?Glu?Ile?Arg?Asp?Leu?Leu?Tyr?Ile?Gly?Lys?Asp?Arg?Lys

405?????????????????410?????????????????415

Asn?Pro?Arg?Glu?Asp?Tyr?Leu?Asp?Val?Tyr?Val?Phe?Gly?Val?Gly?Pro

420?????????????????425?????????????????430

Leu?Val?Asn?Gln?Val?Asn?Ile?Asn?Ala?Leu?Ala?Ser?Lys?Lys?Asp?Asn

435?????????????????440?????????????????445

Glu?Gln?His?Val?Phe?Lys?Val?Lys?Asp?Met?Glu?Asn?Leu?Glu?Asp?Val

450?????????????????455?????????????????460

Phe?Tyr?Gln?Met?Ile?Asp?Glu?Ser?Gln?Ser?Leu?Ser?Leu?Cys?Gly?Met

465?????????????????470?????????????????475?????????????????480

Val?Trp?Glu?His?Arg?Lys?Gly?Thr?Asp?Tyr?His?Lys?Gln?Pro?Trp?Gln

485?????????????????490?????????????????495

Ala?Lys?Ile?Ser?Val?Ile?Arg?Pro?Ser?Lys?Gly?His?Glu?Ser?Cys?Met

500?????????????????505?????????????????510

Gly?Ala?Val?Val?Ser?Glu?Tyr?Phe?Val?Leu?Thr?Ala?Ala?His?Cys?Phe

515?????????????????520?????????????????525

Thr?Val?Asp?Asp?Lys?Glu?His?Ser?Ile?Lys?Val?Ser?Val?Gly?Gly?Glu

530?????????????????535?????????????????540

Lys?Arg?Asp?Leu?Glu?Ile?Glu?Val?Val?Leu?Phe?His?Pro?Asn?Tyr?Asn

545?????????????????550?????????????????555?????????????????560

Ile?Asn?Gly?Lys?Lys?Glu?Ala?Gly?Ile?Pro?Glu?Phe?Tyr?Asp?Tyr?Asp

565?????????????????570?????????????????575

Val?Ala?Leu?Ile?Lys?Leu?Lys?Asn?Lys?Leu?Lys?Tyr?Gly?Gln?Thr?Ile

580?????????????????585?????????????????590

Arg?Pro?Ile?Cys?Leu?Pro?Cys?Thr?Glu?Gly?Thr?Thr?Arg?Ala?Leu?Arg

595?????????????????600?????????????????605

Leu?Pro?Pro?Thr?Thr?Thr?Cys?Gln?Gln?Gln?Lys?Glu?Glu?Leu?Leu?Pro

610?????????????????615?????????????????620

Ala?Gln?Asp?Ile?Lys?Ala?Leu?Phe?Val?Ser?Glu?Glu?Glu?Lys?Lys?Leu

625?????????????????630?????????????????635?????????????????640

Thr?Arg?Lys?Glu?Val?Tyr?Ile?Lys?Asn?Gly?Asp?Lys?Lys?Gly?Ser?Cys

645?????????????????650?????????????????655

Glu?Arg?Asp?Ala?Gln?Tyr?Ala?Pro?Gly?Tyr?Asp?Lys?Val?Lys?Asp?Ile

660?????????????????665?????????????????670

Ser?Glu?Val?Val?Thr?Pro?Arg?Phe?Leu?Cys?Thr?Gly?Gly?Val?Ser?Pro

675?????????????????680?????????????????685

Tyr?Ala?Asp?Pro?Asn?Thr?Cys?Arg?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ile

690?????????????????695?????????????????700

Val?His?Lys?Arg?Ser?Arg?Phe?Ile?Gln?Val?Gly?Val?Ile?Ser?Trp?Gly

705?????????????????710?????????????????715?????????????????720

Val?Val?Asp?Val?Cys?Lys?Asn?Gln?Lys?Arg?Gln?Lys?Gln?Val?Pro?Ala

725?????????????????730?????????????????735

His?Ala?Arg?Asp?Phe?His?Ile?Asn?Leu?Phe?Gln?Val?Leu?Pro?Trp?Leu

740?????????????????745?????????????????750

Lys?Glu?Lys?Leu?Gln?Asp?Glu?Asp?Leu?Gly?Phe?Leu

755?????????????????760

<210>9

<211>2428

<212>DNA

＜213〉mouse kind (Mus sp.)

<400>9

tgtagccaga?tccagcattt?gggtttcagt?ttggacagga?ggtcaaatag?gcacccagag????60

tgacctggag?agggctttgg?gccactggac?tctctggtgc?tttccatgac?aatggagagc???120

ccccagctct?gcctcgtcct?cttggtctta?ggcttctcct?ctggaggtgt?gagcgcaact???180

ccagtgcttg?aggcccggcc?ccaagtctcc?tgctctctgg?agggagtaga?gatcaaaggc???240

ggctcctttc?aacttctcca?aggcggtcag?gccctggagt?acctatgtcc?ctctggcttc???300

tacccatacc?ccgtgcagac?tcgaacctgc?agatccacag?gctcctggag?cgacctgcag???360

acccgagacc?aaaagattgt?ccagaaggcg?gaatgcagag?caatacgctg?cccacgaccg???420

caggactttg?aaaatgggga?attctggccc?cggtccccct?tctacaacct?gagtgaccag???480

atttcttttc?aatgctatga?tggttacgtt?ctccggggct?ctgctaatcg?cacctgccaa???540

gagaatggcc?ggtgggatgg?gcaaacagca?atttgtgatg?atggagctgg?atactgtccc???600

aatcccggta?ttcctattgg?gacaaggaag?gtgggtagcc?aataccgcct?tgaagacatt???660

gttacttacc?actgcagccg?gggacttgtc?ctgcgtggct?cccagaagcg?aaagtgtcaa???720

gaaggtggct?catggagtgg?gacagagcct?tcctgccaag?attccttcat?gtatgacagc???780

cctcaagaag?tggccgaagc?attcctatcc?tccctgacag?agaccatcga?aggagccgat???840

gctgaggatg?ggcacagccc?aggagaacag?cagaagagga?agattgtcct?agacccctcg???900

ggctccatga?atatctacct?ggtgctagat?ggatcagaca?gcatcggaag?cagcaacttc???960

acaggggcta?agcggtgcct?caccaacttg?attgagaagg?tggcgagtta?cggggtgagg??1020

ccacgatatg?gtctcctgac?atatgctaca?gtccccaaag?tgttggtcag?agtgtctgat??1080

gagaggagta?gcgatgccga?ctgggtcaca?gagaagctca?accaaatcag?ttatgaagac??1140

cacaagctga?agtcagggac?caacaccaag?agggctctcc?aggctgtgta?tagcatgatg??1200

agctgggcag?gggatgcccc?gcctgaaggc?tggaatagaa?cccgccatgt?catcatcatt??1260

atgactgatg?gcttgcacaa?catgggtgga?aaccctgtca?ctgtcattca?ggacatccga??1320

gccttgctgg?acatcggcag?ggatcccaaa?aatcccaggg?aggattacct?ggatgtgtat??1380

gtgtttgggg?tcgggcctct?ggtggactcc?gtgaacatca?atgccttagc?ttccaaaaag??1440

gacaatgagc?atcatgtgtt?taaagtcaag?gatatggaag?acctggagaa?tgttttctac??1500

caaatgattg?atgaaaccaa?atctctgagt?ctctgtggca?tggtgtggga?gcataaaaaa??1560

ggcaacgatt?atcataagca?accatggcaa?gccaagatct?cagtcactcg?ccctctgaaa??1620

ggacatgaga?cctgtatggg?ggccgtggtg?tctgagtact?tcgtgctgac?agcagcgcac??1680

tgcttcatgg?tggatgatca?gaaacattcc?atcaaggtca?gcgtgggggg?tcagaggcgg??1740

gacctggaga?ttgaagaggt?cctgttccac?cccaaataca?atattaatgg?gaaaaaggca??1800

gaagggatcc?ctgagttcta?tgattatgat?gtggccctag?tcaagctcaa?gaacaagctc??1860

aagtatggcc?agactctcag?gcccatctgt?ctcccctgca?cggagggaac?cacacgagcc??1920

ttgaggcttc?ctcagacagc?cacctgcaag?cagcacaagg?aacagttgct?ccctgtgaag??1980

gatgtcaaag?ctctgtttgt?atctgagcaa?gggaagagcc?tgactcggaa?ggaggtgtac??2040

atcaagaatg?gggacaagaa?agccagttgt?gagagagatg?ctacaaaggc?ccaaggctat??2100

gagaaggtca?aagatgcctc?tgaggtggtc?actccacggt?tcctctgcac?aggaggggtg??2160

gatccctatg?ctgaccccaa?cacatgcaaa?ggagattccg?ggggccctct?cattgttcac??2220

aagagaagcc?gcttcattca?agttggtgtg?attagctggg?gagtagtaga?tgtctgcaga??2280

gaccagaggc?ggcaacagct?ggtaccctct?tatgcccgga?acttccacat?caacctcttc??2340

caggtgctgc?cctggctaaa?ggacaagctc?aaagatgagg?atttgggttt?tctatgaaga??2400

gcttcctgca?gggagagtgt?gaggacag?????????????????????????????????????2428

<210>10

<211>761

<212>PRT

＜213〉mouse kind (Mus sp.)

<400>10

Met?Glu?Ser?Pro?Gln?Leu?Cys?Leu?Val?Leu?Leu?Val?Leu?Gly?Phe?Ser

1???????????????5???????????????????10??????????????????15

Ser?Gly?Gly?Val?Ser?Ala?Thr?Pro?Val?Leu?Glu?Ala?Arg?Pro?Gln?Val

20??????????????????25??????????????????30

Ser?Cys?Ser?Leu?Glu?Gly?Val?Glu?Ile?Lys?Gly?Gly?Ser?Phe?Gln?Leu

35??????????????????40??????????????????45

Leu?Gln?Gly?Gly?Gln?Ala?Leu?Glu?Tyr?Leu?Cys?Pro?Ser?Gly?Phe?Tyr

50??????????????????55??????????????????60

Pro?Tyr?Pro?Val?Gln?Thr?Arg?Thr?Cys?Arg?Ser?Thr?Gly?Ser?Trp?Ser

65??????????????????70??????????????????75??????????????????80

Asp?Leu?Gln?Thr?Arg?Asp?Gln?Lys?Ile?Val?Gln?Lys?Ala?Glu?Cys?Arg

85??????????????????90??????????????????95

Ala?Ile?Arg?Cys?Pro?Arg?Pro?Gln?Asp?Phe?Glu?Asn?Gly?Glu?Phe?Trp

100?????????????????105?????????????????110

Pro?Arg?Ser?Pro?Phe?Tyr?Asn?Leu?Ser?Asp?Gln?Ile?Ser?Phe?Gln?Cys

115?????????????????120?????????????????125

Tyr?Asp?Gly?Tyr?Val?Leu?Arg?Gly?Ser?Ala?Asn?Arg?Thr?Cys?Gln?Glu

130?????????????????135?????????????????140

Asn?Gly?Arg?Trp?Asp?Gly?Gln?Thr?Ala?Ile?Cys?Asp?Asp?Gly?Ala?Gly

145?????????????????150?????????????????155?????????????????160

Tyr?Cys?Pro?Asn?Pro?Gly?Ile?Pro?Ile?Gly?Thr?Arg?Lys?Val?Gly?Ser

165?????????????????170?????????????????175

Gln?Tyr?Arg?Leu?Glu?Asp?Ile?Val?Thr?Tyr?His?Cys?Ser?Arg?Gly?Leu

180?????????????????185?????????????????190

Val?Leu?Arg?Gly?Ser?Gln?Lys?Arg?Lys?Cys?Gln?Glu?Gly?Gly?Ser?Trp

195?????????????????200?????????????????205

Ser?Gly?Thr?Glu?Pro?Ser?Cys?Gln?Asp?Ser?Phe?Met?Tyr?Asp?Ser?Pro

210?????????????????215?????????????????220

Gln?Glu?Val?Ala?Glu?Ala?Phe?Leu?Ser?Ser?Leu?Thr?Glu?Thr?Ile?Glu

225?????????????????230?????????????????235?????????????????240

Gly?Ala?Asp?Ala?Glu?Asp?Gly?His?Ser?Pro?Gly?Glu?Gln?Gln?Lys?Arg

245?????????????????250?????????????????255

Lys?Ile?Val?Leu?Asp?Pro?Ser?Gly?Ser?Met?Asn?Ile?Tyr?Leu?Val?Leu

260?????????????????265?????????????????270

Asp?Gly?Ser?Asp?Ser?Ile?Gly?Ser?Ser?Asn?Phe?Thr?Gly?Ala?Lys?Arg

275?????????????????280?????????????????285

Cys?Leu?Thr?Asn?Leu?Ile?Glu?Lys?Val?Ala?Ser?Tyr?Gly?Val?Arg?Pro

290?????????????????295?????????????????300

Arg?Tyr?Gly?Leu?Leu?Thr?Tyr?Ala?Thr?Val?Pro?Lys?Val?Leu?Val?Arg

305?????????????????310?????????????????315?????????????????320

Val?Ser?Asp?Glu?Arg?Ser?Ser?Asp?Ala?Asp?Trp?Val?Thr?Glu?Lys?Leu

325?????????????????330?????????????????335

Asn?Gln?Ile?Ser?Tyr?Glu?Asp?His?Lys?Leu?Lys?Ser?Gly?Thr?Asn?Thr

340?????????????????345?????????????????350

Lys?Arg?Ala?Leu?Gln?Ala?Val?Tyr?Ser?Met?Met?Ser?Trp?Ala?Gly?Asp

355?????????????????360?????????????????365

Ala?Pro?Pro?Glu?Gly?Trp?Asn?Arg?Thr?Arg?His?Val?Ile?Ile?Ile?Met

370?????????????????375?????????????????380

Thr?Asp?Gly?Leu?His?Asn?Met?Gly?Gly?Asn?Pro?Val?Thr?Val?Ile?Gln

385?????????????????390?????????????????395?????????????????400

Asp?Ile?Arg?Ala?Leu?Leu?Asp?Ile?Gly?Arg?Asp?Pro?Lys?Asn?Pro?Arg

405?????????????????410?????????????????415

Glu?Asp?Tyr?Leu?Asp?Val?Tyr?Val?Phe?Gly?Val?Gly?Pro?Leu?Val?Asp

420?????????????????425?????????????????430

Ser?Val?Asn?Ile?Asn?Ala?Leu?Ala?Ser?Lys?Lys?Asp?Asn?Glu?His?His

435?????????????????440?????????????????445

Val?Phe?Lys?Val?Lys?Asp?Met?Glu?Asp?Leu?Glu?Asn?Val?Phe?Tyr?Gln

450?????????????????455?????????????????460

Met?Ile?Asp?Glu?Thr?Lys?Ser?Leu?Ser?Leu?Cys?Gly?Met?Val?Trp?Glu

465?????????????????470?????????????????475?????????????????480

His?Lys?Lys?Gly?Asn?Asp?Tyr?His?Lys?Gln?Pro?Trp?Gln?Ala?Lys?Ile

485?????????????????490?????????????????495

Ser?Val?Thr?Arg?Pro?Leu?Lys?Gly?His?Glu?Thr?Cys?Met?Gly?Ala?Val

500?????????????????505?????????????????510

Val?Ser?Glu?Tyr?Phe?Val?Leu?Thr?Ala?Ala?His?Cys?Phe?Met?Val?Asp

515?????????????????520?????????????????525

Asp?Gln?Lys?His?Ser?Ile?Lys?Val?Ser?Val?Gly?Gly?Gln?Arg?Arg?Asp

530?????????????????535?????????????????540

Leu?Glu?Ile?Glu?Glu?Val?Leu?Phe?His?Pro?Lys?Tyr?Asn?Ile?Asn?Gly

545?????????????????550?????????????????555?????????????????560

Lys?Lys?Ala?Glu?Gly?Ile?Pro?Glu?Phe?Tyr?Asp?Tyr?Asp?Val?Ala?Leu

565?????????????????570?????????????????575

Val?Lys?Leu?Lys?Asn?Lys?Leu?Lys?Tyr?Gly?Gln?Thr?Leu?Arg?Pro?Ile

580?????????????????585?????????????????590

Cys?Leu?Pro?Cys?Thr?Glu?Gly?Thr?Thr?Arg?Ala?Leu?Arg?Leu?Pro?Gln

595?????????????????600?????????????????605

Thr?Ala?Thr?Cys?Lys?Gln?His?Lys?Glu?Gln?Leu?Leu?Pro?Val?Lys?Asp

610?????????????????615?????????????????620

Val?Lys?Ala?Leu?Phe?Val?Ser?Glu?Gln?Gly?Lys?Ser?Leu?Thr?Arg?Lys

625?????????????????630?????????????????635?????????????????640

Glu?Val?Tyr?Ile?Lys?Asn?Gly?Asp?Lys?Lys?Ala?Ser?Cys?Glu?Arg?Asp

645?????????????????650?????????????????655

Ala?Thr?Lys?Ala?Gln?Gly?Tyr?Glu?Lys?Val?Lys?Asp?Ala?Ser?Glu?Val

660?????????????????665?????????????????670

Val?Thr?Pro?Arg?Phe?Leu?Cys?Thr?Gly?Gly?Val?Asp?Pro?Tyr?Ala?Asp

675?????????????????680?????????????????685

Pro?Asn?Thr?Cys?Lys?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ile?Val?His?Lys

690?????????????????695?????????????????700

Arg?Ser?Arg?Phe?Ile?Gln?Val?Gly?Val?Ile?Ser?Trp?Gly?Val?Val?Asp

705?????????????????710?????????????????715?????????????????720

Val?Cys?Arg?Asp?Gln?Arg?Arg?Gln?Gln?Leu?Val?Pro?Ser?Tyr?Ala?Arg

725?????????????????730?????????????????735

Asn?Phe?His?Ile?Asn?Leu?Phe?Gln?Val?Leu?Pro?Trp?Leu?Lys?Asp?Lys

740?????????????????745?????????????????750

Leu?Lys?Asp?Glu?Asp?Leu?Gly?Phe?Leu

755?????????????????760

<210>11

<211>2428

<212>DNA

＜213〉mouse kind (Mus sp.)

<400>11

tgtagccaga?tccagcattt?gggtttcagt?ttggacagga?ggtcaaatag?gcacccagag????60

tgacctggag?agggctttgg?gccactggac?tctctggtgc?tttccatgac?aatggagagc???120

ccccagctct?gcctcgtcct?cttggtctta?ggcttctcct?ctggaggtgt?gagcgcaact???180

ccagtgcttg?aggcccggcc?ccaagtctcc?tgctctctgg?agggagtaga?gatcaaaggc???240

ggctcctttc?aacttctcca?aggcggtcag?gccctggagt?acctatgtcc?ctctggcttc???300

tacccatacc?ccgtgcagac?tcgaacctgc?agatccacag?gctcctggag?cgacctgcag???360

acccgagacc?aaaagattgt?ccagaaggcg?gaatgcagag?caatacgctg?cccacgaccg???420

caggactttg?aaaatgggga?attctggccc?cggtccccct?tctacaacct?gagtgaccag???480

atttcttttc?aatgctatga?tggttacgtt?ctccggggct?ctgctaatcg?cacctgccaa???540

gagaatggcc?ggtgggatgg?gcaaacagca?atttgtgatg?atggagctgg?atactgtccc???600

aatcccggta?ttcctattgg?gacaaggaag?gtgggtagcc?aataccgcct?tgaagacatt???660

gttacttacc?actgcagccg?gggacttgtc?ctgcgtggct?cccagaagcg?aaagtgtcaa???720

gaaggtggct?catggagtgg?gacagagcct?tcctgccaag?attccttcat?gtatgacagc???780

cctcaagaag?tggccgaagc?attcctatcc?tccctgacag?agaccatcga?aggagccgat???840

gctgaggatg?ggcacagccc?aggagaacag?cagaagagga?agattgtcct?agacccctcg???900

ggctccatga?atatctacct?ggtgctagat?ggatcaggca?gcatcggaag?cagcgacttc???960

acaggggcta?agcggtgcct?caccaacttg?attgagaagg?tggcgagtta?cggggtgagg??1020

ccacgatatg?gtctcctgac?atatgctaca?gtccccaaag?tgttggtcag?agtgtctgat??1080

gagaggagta?gcgatgccga?ctgggtcaca?gagaagctca?accaaatcag?ttatgaagac??1140

cacaagctga?agtcagggac?caacaccaag?agggctctcc?aggctgtgta?tagcatgatg??1200

agctgggcag?gggatgcccc?gcctgaaggc?tggaatagaa?cccgccatgt?catcatcatt??1260

atgactgatg?gcttgcacaa?catgggtgga?aaccctgtca?ctgtcattca?ggacatccga??1320

gccttgctgg?acatcggcag?ggatcccaaa?aatcccaggg?aggattacct?ggatgtgtat??1380

gtgtttgggg?tcgggcctct?ggtggactcc?gtgaacatca?atgccttagc?ttccaaaaag??1440

gacaatgagc?atcatgtgtt?taaagtcaag?gatatggaag?acctggagaa?tgttttctac??1500

caaatgattg?atgaaaccaa?atctctgagt?ctctgtggca?tggtgtggga?gcataaaaaa??1560

ggcaacgatt?atcataagca?accatggcaa?gccaagatct?cagtcactcg?ccctctgaaa??1620

ggacatgaga?cctgtatggg?ggccgtggtg?tctgagtact?tcgtgctgac?agcagcgcac??1680

tgcttcatgg?tggatgatca?gaaacattcc?atcaaggtca?gcgtgggggg?tcagaggcgg??1740

gacctggaga?ttgaagaggt?cctgttccac?cccaaataca?atattaatgg?gaaaaaggca??1800

gaagggatcc?ctgagttcta?tgattatgat?gtggccctag?tcaagctcaa?gaacaagctc??1860

aagtatggcc?agactctcag?gcccatctgt?ctcccctgca?cggagggaac?cacacgagcc??1920

ttgaggcttc?ctcagacagc?cacctgcaag?cagcacaagg?aacagttgct?ccctgtgaag??1980

gatgtcaaag?ctctgtttgt?atctgagcaa?gggaagagcc?tgactcggaa?ggaggtgtac??2040

atcaagaatg?gggacaagaa?agccagttgt?gagagagatg?ctacaaaggc?ccaaggctat??2100

gagaaggtca?aagatgcctc?tgaggtggtc?actccacggt?tcctctgcac?aggaggggtg??2160

gatccctatg?ctgaccccaa?cacatgcaaa?ggagattccg?ggggccctct?cattgttcac??2220

aagagaagcc?gcttcattca?agttggtgtg?attagctggg?gagtagtaga?tgtctgcaga??2280

gaccagaggc?ggcaacagct?ggtaccctct?tatgcccgga?acttccacat?caacctcttc??2340

caggtgctgc?cctggctaaa?ggacaagctc?aaagatgagg?atttgggttt?tctatgaaga??2400

gcttcctgca?gggagagtgt?gaggacag?????????????????????????????????????2428

<210>12

<211>761

<212>PRT

＜213〉mouse kind (Mus sp.)

<400>12

Met?Glu?Ser?Pro?Gln?Leu?Cys?Leu?Val?Leu?Leu?Val?Leu?Gly?Phe?Ser

1???????????????5???????????????????10??????????????????15

Ser?Gly?Gly?Val?Ser?Ala?Thr?Pro?Val?Leu?Glu?Ala?Arg?Pro?Gln?Val

20??????????????????25??????????????????30

Ser?Cys?Ser?Leu?Glu?Gly?Val?Glu?Ile?Lys?Gly?Gly?Ser?Phe?Gln?Leu

35??????????????????40??????????????????45

Leu?Gln?Gly?Gly?Gln?Ala?Leu?Glu?Tyr?Leu?Cys?Pro?Ser?Gly?Phe?Tyr

50??????????????????55??????????????????60

Pro?Tyr?Pro?Val?Gln?Thr?Arg?Thr?Cys?Arg?Ser?Thr?Gly?Ser?Trp?Ser

65??????????????????70??????????????????75??????????????????80

Asp?Leu?Gln?Thr?Arg?Asp?Gln?Lys?Ile?Val?Gln?Lys?Ala?Glu?Cys?Arg

85??????????????????90??????????????????95

Ala?Ile?Arg?Cys?Pro?Arg?Pro?Gln?Asp?Phe?Glu?Asn?Gly?Glu?Phe?Trp

100?????????????????105?????????????????110

Pro?Arg?Ser?Pro?Phe?Tyr?Asn?Leu?Ser?Asp?Gln?Ile?Ser?Phe?Gln?Cys

115?????????????????120?????????????????125

Tyr?Asp?Gly?Tyr?Val?Leu?Arg?Gly?Ser?Ala?Asn?Arg?Thr?Cys?Gln?Glu

130?????????????????135?????????????????140

Asn?Gly?Arg?Trp?Asp?Gly?Gln?Thr?Ala?Ile?Cys?Asp?Asp?Gly?Ala?Gly

145?????????????????150?????????????????155?????????????????160

Tyr?Cys?Pro?Asn?Pro?Gly?Ile?Pro?Ile?Gly?Thr?Arg?Lys?Val?Gly?Ser

165?????????????????170?????????????????175

Gln?Tyr?Arg?Leu?Glu?Asp?Ile?Val?Thr?Tyr?His?Cys?Ser?Arg?Gly?Leu

180?????????????????185?????????????????190

Val?Leu?Arg?Gly?Ser?Gln?Lys?Arg?Lys?Cys?Gln?Glu?Gly?Gly?Ser?Trp

195?????????????????200?????????????????205

Ser?Gly?Thr?Glu?Pro?Ser?Cys?Gln?Asp?Ser?Phe?Met?Tyr?Asp?Ser?Pro

210?????????????????215?????????????????220

Gln?Glu?Val?Ala?Glu?Ala?Phe?Leu?Ser?Ser?Leu?Thr?Glu?Thr?Ile?Glu

225?????????????????230?????????????????235?????????????????240

Gly?Ala?Asp?Ala?Glu?Asp?Gly?His?Ser?Pro?Gly?Glu?Gln?Gln?Lys?Arg

245?????????????????250?????????????????255

Lys?Ile?Val?Leu?Asp?Pro?Ser?Gly?Ser?Met?Asn?Ile?Tyr?Leu?Val?Leu

260?????????????????265?????????????????270

Asp?Gly?Ser?Gly?Ser?Ile?Gly?Ser?Ser?Asp?Phe?Thr?Gly?Ala?Lys?Arg

275?????????????????280?????????????????285

Cys?Leu?Thr?Asn?Leu?Ile?Glu?Lys?Val?Ala?Ser?Tyr?Gly?Val?Arg?Pro

290?????????????????295?????????????????300

Arg?Tyr?Gly?Leu?Leu?Thr?Tyr?Ala?Thr?Val?Pro?Lys?Val?Leu?Val?Arg

305?????????????????310?????????????????315?????????????????320

Val?Ser?Asp?Glu?Arg?Ser?Ser?Asp?Ala?Asp?Trp?Val?Thr?Glu?Lys?Leu

325?????????????????330?????????????????335

Asn?Gln?Ile?Ser?Tyr?Glu?Asp?His?Lys?Leu?Lys?Ser?Gly?Thr?Asn?Thr

340?????????????????345?????????????????350

Lys?Arg?Ala?Leu?Gln?Ala?Val?Tyr?Ser?Met?Met?Ser?Trp?Ala?Gly?Asp

355?????????????????360?????????????????365

Ala?Pro?Pro?Glu?Gly?Trp?Asn?Arg?Thr?Arg?His?Val?Ile?Ile?Ile?Met

370?????????????????375?????????????????380

Thr?Asp?Gly?Leu?His?Asn?Met?Gly?Gly?Asn?Pro?Val?Thr?Val?Ile?Gln

385?????????????????390?????????????????395?????????????????400

Asp?Ile?Arg?Ala?Leu?Leu?Asp?Ile?Gly?Arg?Asp?Pro?Lys?Asn?Pro?Arg

405?????????????????410?????????????????415

Glu?Asp?Tyr?Leu?Asp?Val?Tyr?Val?Phe?Gly?Val?Gly?Pro?Leu?Val?Asp

420?????????????????425?????????????????430

Ser?Val?Asn?Ile?Asn?Ala?Leu?Ala?Ser?Lys?Lys?Asp?Asn?Glu?His?His

435?????????????????440?????????????????445

Val?Phe?Lys?Val?Lys?Asp?Met?Glu?Asp?Leu?Glu?Asn?Val?Phe?Tyr?Gln

450?????????????????455?????????????????460

Met?Ile?Asp?Glu?Thr?Lys?Ser?Leu?Ser?Leu?Cys?Gly?Met?Val?Trp?Glu

465?????????????????470?????????????????475?????????????????480

His?Lys?Lys?Gly?Asn?Asp?Tyr?His?Lys?Gln?Pro?Trp?Gln?Ala?Lys?Ile

485?????????????????490?????????????????495

Ser?Val?Thr?Arg?Pro?Leu?Lys?Gly?His?Glu?Thr?Cys?Met?Gly?Ala?Val

500?????????????????505?????????????????510

Val?Ser?Glu?Tyr?Phe?Val?Leu?Thr?Ala?Ala?His?Cys?Phe?Met?Val?Asp

515?????????????????520?????????????????525

Asp?Gln?Lys?His?Ser?Ile?Lys?Val?Ser?Val?Gly?Gly?Gln?Arg?Arg?Asp

530?????????????????535?????????????????540

Leu?Glu?Ile?Glu?Glu?Val?Leu?Phe?His?Pro?Lys?Tyr?Asn?Ile?Asn?Gly

545?????????????????550?????????????????555?????????????????560

Lys?Lys?Ala?Glu?Gly?Ile?Pro?Glu?Phe?Tyr?Asp?Tyr?Asp?Val?Ala?Leu

565?????????????????570?????????????????575

Val?Lys?Leu?Lys?Asn?Lys?Leu?Lys?Tyr?Gly?Gln?Thr?Leu?Arg?Pro?Ile

580?????????????????585?????????????????590

Cys?Leu?Pro?Cys?Thr?Glu?Gly?Thr?Thr?Arg?Ala?Leu?Arg?Leu?Pro?Gln

595?????????????????600?????????????????605

Thr?Ala?Thr?Cys?Lys?Gln?His?Lys?Glu?Gln?Leu?Leu?Pro?Val?Lys?Asp

610?????????????????615?????????????????620

Val?Lys?Ala?Leu?Phe?Val?Ser?Glu?Gln?Gly?Lys?Ser?Leu?Thr?Arg?Lys

625?????????????????630?????????????????635?????????????????640

Glu?Val?Tyr?Ile?Lys?Asn?Gly?Asp?Lys?Lys?Ala?Ser?Cys?Glu?Arg?Asp

645?????????????????650?????????????????655

Ala?Thr?Lys?Ala?Gln?Gly?Tyr?Glu?Lys?Val?Lys?Asp?Ala?Ser?Glu?Val

660?????????????????665?????????????????670

Val?Thr?Pro?Arg?Phe?Leu?Cys?Thr?Gly?Gly?Val?Asp?Pro?Tyr?Ala?Asp

675?????????????????680?????????????????685

Pro?Asn?Thr?Cys?Lys?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ile?Val?His?Lys

690?????????????????695?????????????????700

Arg?Ser?Arg?Phe?Ile?Gln?Val?Gly?Val?Ile?Ser?Trp?Gly?Val?Val?Asp

705?????????????????710?????????????????715?????????????????720

Val?Cys?Arg?Asp?Gln?Arg?Arg?Gln?Gln?Leu?Val?Pro?Ser?Tyr?Ala?Arg

725?????????????????730?????????????????735

Asn?Phe?His?Ile?Asn?Leu?Phe?Gln?Val?Leu?Pro?Trp?Leu?Lys?Asp?Lys

740?????????????????745?????????????????750

Leu?Lys?Asp?Glu?Asp?Leu?Gly?Phe?Leu

755?????????????????760

<210>13

<211>2428

<212>DNA

＜213〉mouse kind (Mus sp.)

<400>13

tgtagccaga?tccagcattt?gggtttcagt?ttggacagga?ggtcaaatag?gcacccagag????60

tgacctggag?agggctttgg?gccactggac?tctctggtgc?tttccatgac?aatggagagc???120

ccccagctct?gcctcgtcct?cttggtctta?ggcttctcct?ctggaggtgt?gagcgcaact???180

ccagtgcttg?aggcccggcc?ccaagtctcc?tgctctctgg?agggagtaga?gatcaaaggc???240

ggctcctttc?aacttctcca?aggcggtcag?gccctggagt?acctatgtcc?ctctggcttc???300

tacccatacc?ccgtgcagac?tcgaacctgc?agatccacag?gctcctggag?cgacctgcag???360

acccgagacc?aaaagattgt?ccagaaggcg?gaatgcagag?caatacgctg?cccacgaccg???420

caggactttg?aaaatgggga?attctggccc?cggtccccct?tctacaacct?gagtgaccag???480

atttcttttc?aatgctatga?tggttacgtt?ctccggggct?ctgctaatcg?cacctgccaa???540

gagaatggcc?ggtgggatgg?gcaaacagca?atttgtgatg?atggagctgg?atactgtccc???600

aatcccggta?ttcctattgg?gacaaggaag?gtgggtagcc?aataccgcct?tgaagacatt???660

gttacttacc?actgcagccg?gggacttgtc?ctgcgtggct?cccagaagcg?aaagtgtcaa???720

gaaggtggct?catggagtgg?gacagagcct?tcctgccaag?attccttcat?gtatgacagc???780

cctcaagaag?tggccgaagc?attcctatcc?tccctgacag?agaccatcga?aggagccgat???840

gctgaggatg?ggcacagccc?aggagaacag?caggcggcag?cgattgtcct?agacccctcg???900

ggctccatga?atatctacct?ggtgctagat?ggatcaggca?gcatcggaag?cagcgacttc???960

acaggggcta?agcggtgcct?caccaacttg?attgagaagg?tggcgagtta?cggggtgagg??1020

ccacgatatg?gtctcctgac?atatgctaca?gtccccaaag?tgttggtcag?agtgtctgat??1080

gagaggagta?gcgatgccga?ctgggtcaca?gagaagctca?accaaatcag?ttatgaagac??1140

cacaagctga?agtcagggac?caacaccaag?agggctctcc?aggctgtgta?tagcatgatg??1200

agctgggcag?gggatgcccc?gcctgaaggc?tggaatagaa?cccgccatgt?catcatcatt??1260

atgactgatg?gcttgcacaa?catgggtgga?aaccctgtca?ctgtcattca?ggacatccga??1320

gccttgctgg?acatcggcag?ggatcccaaa?aatcccaggg?aggattacct?ggatgtgtat??1380

gtgtttgggg?tcgggcctct?ggtggactcc?gtgaacatca?atgccttagc?ttccaaaaag??1440

gacaatgagc?atcatgtgtt?taaagtcaag?gatatggaag?acctggagaa?tgttttctac??1500

caaatgattg?atgaaaccaa?atctctgagt?ctctgtggca?tggtgtggga?gcataaaaaa??1560

ggcaacgatt?atcataagca?accatggcaa?gccaagatct?cagtcactcg?ccctctgaaa??1620

ggacatgaga?cctgtatggg?ggccgtggtg?tctgagtact?tcgtgctgac?agcagcgcac??1680

tgcttcatgg?tggatgatca?gaaacattcc?atcaaggtca?gcgtgggggg?tcagaggcgg??1740

gacctggaga?ttgaagaggt?cctgttccac?cccaaataca?atattaatgg?gaaaaaggca??1800

gaagggatcc?ctgagttcta?tgattatgat?gtggccctag?tcaagctcaa?gaacaagctc??1860

aagtatggcc?agactctcag?gcccatctgt?ctcccctgca?cggagggaac?cacacgagcc??1920

ttgaggcttc?ctcagacagc?cacctgcaag?cagcacaagg?aacagttgct?ccctgtgaag??1980

gatgtcaaag?ctctgtttgt?atctgagcaa?gggaagagcc?tgactcggaa?ggaggtgtac??2040

atcaagaatg?gggacaagaa?agccagttgt?gagagagatg?ctacaaaggc?ccaaggctat??2100

gagaaggtca?aagatgcctc?tgaggtggtc?actccacggt?tcctctgcac?aggaggggtg??2160

gatccctatg?ctgaccccaa?cacatgcaaa?ggagattccg?ggggccctct?cattgttcac??2220

aagagaagcc?gcttcattca?agttggtgtg?attagctggg?gagtagtaga?tgtctgcaga??2280

gaccagaggc?ggcaacagct?ggtaccctct?tatgcccggg?acttccacat?caacctcttc??2340

caggtgctgc?cctggctaaa?ggacaagctc?aaagatgagg?atttgggttt?tctatgaaga??2400

gcttcctgca?gggagagtgt?gaggacag?????????????????????????????????????2428

<210>14

<211>761

<212>PRT

＜213〉mouse kind (Mus sp.)

<400>14

Met?Glu?Ser?Pro?Gln?Leu?Cys?Leu?Val?Leu?Leu?Val?Leu?Gly?Phe?Ser

1???????????????5???????????????????10??????????????????15

Ser?Gly?Gly?Val?Ser?Ala?Thr?Pro?Val?Leu?Glu?Ala?Arg?Pro?Gln?Val

20??????????????????25??????????????????30

Ser?Cys?Ser?Leu?Glu?Gly?Val?Glu?Ile?Lys?Gly?Gly?Ser?Phe?Gln?Leu

35??????????????????40??????????????????45

Leu?Gln?Gly?Gly?Gln?Ala?Leu?Glu?Tyr?Leu?Cys?Pro?Ser?Gly?Phe?Tyr

50??????????????????55??????????????????60

Pro?Tyr?Pro?Val?Gln?Thr?Arg?Thr?Cys?Arg?Ser?Thr?Gly?Ser?Trp?Ser

65??????????????????70??????????????????75??????????????????80

Asp?Leu?Gln?Thr?Arg?Asp?Gln?Lys?Ile?Val?Gln?Lys?Ala?Glu?Cys?Arg

85??????????????????90??????????????????95

Ala?Ile?Arg?Cys?Pro?Arg?Pro?Gln?Asp?Phe?Glu?Asn?Gly?Glu?Phe?Trp

100?????????????????105?????????????????110

Pro?Arg?Ser?Pro?Phe?Tyr?Asn?Leu?Ser?Asp?Gln?Ile?Ser?Phe?Gln?Cys

115?????????????????120?????????????????125

Tyr?Asp?Gly?Tyr?Val?Leu?Arg?Gly?Ser?Ala?Asn?Arg?Thr?Cys?Gln?Glu

130?????????????????135?????????????????140

Asn?Gly?Arg?Trp?Asp?Gly?Gln?Thr?Ala?Ile?Cys?Asp?Asp?Gly?Ala?Gly

145?????????????????150?????????????????155?????????????????160

Tyr?Cys?Pro?Asn?Pro?Gly?Ile?Pro?Ile?Gly?Thr?Arg?Lys?Val?Gly?Ser

165?????????????????170?????????????????175

Gln?Tyr?Arg?Leu?Glu?Asp?Ile?Val?Thr?Tyr?His?Cys?Ser?Arg?Gly?Leu

180?????????????????185?????????????????190

Val?Leu?Arg?Gly?Ser?Gln?Lys?Arg?Lys?Cys?Gln?Glu?Gly?Gly?Ser?Trp

195?????????????????200?????????????????205

Ser?Gly?Thr?Glu?Pro?Ser?Cys?Gln?Asp?Ser?Phe?Met?Tyr?Asp?Ser?Pro

210?????????????????215?????????????????220

Gln?Glu?Val?Ala?Glu?Ala?Phe?Leu?Ser?Ser?Leu?Thr?Glu?Thr?Ile?Glu

225?????????????????230?????????????????235?????????????????240

Gly?Ala?Asp?Ala?Glu?Asp?Gly?His?Ser?Pro?Gly?Glu?Gln?Gln?Ala?Ala

245?????????????????250?????????????????255

Ala?Ile?Val?Leu?Asp?Pro?Ser?Gly?Ser?Met?Asn?Ile?Tyr?Leu?Val?Leu

260?????????????????265?????????????????270

Asp?Gly?Ser?Gly?Ser?Ile?Gly?Ser?Ser?Asp?Phe?Thr?Gly?Ala?Lys?Arg

275?????????????????280?????????????????285

Cys?Leu?Thr?Asn?Leu?Ile?Glu?Lys?Val?Ala?Ser?Tyr?Gly?Val?Arg?Pro

290?????????????????295?????????????????300

Arg?Tyr?Gly?Leu?Leu?Thr?Tyr?Ala?Thr?Val?Pro?Lys?Val?Leu?Val?Arg

305?????????????????310?????????????????315?????????????????320

Val?Ser?Asp?Glu?Arg?Ser?Ser?Asp?Ala?Asp?Trp?Val?Thr?Glu?Lys?Leu

325?????????????????330?????????????????335

Asn?Gln?Ile?Ser?Tyr?Glu?Asp?His?Lys?Leu?Lys?Ser?Gly?Thr?Asn?Thr

340?????????????????345?????????????????350

Lys?Arg?Ala?Leu?Gln?Ala?Val?Tyr?Ser?Met?Met?Ser?Trp?Ala?Gly?Asp

355?????????????????360?????????????????365

Ala?Pro?Pro?Glu?Gly?Trp?Asn?Arg?Thr?Arg?His?Val?Ile?Ile?Ile?Met

370?????????????????375?????????????????380

Thr?Asp?Gly?Leu?His?Asn?Met?Gly?Gly?Asn?Pro?Val?Thr?Val?Ile?Gln

385?????????????????390?????????????????395?????????????????400

Asp?Ile?Arg?Ala?Leu?Leu?Asp?Ile?Gly?Arg?Asp?Pro?Lys?Asn?Pro?Arg

405?????????????????410?????????????????415

Glu?Asp?Tyr?Leu?Asp?Val?Tyr?Val?Phe?Gly?Val?Gly?Pro?Leu?Val?Asp

420?????????????????425?????????????????430

Ser?Val?Asn?Ile?Asn?Ala?Leu?Ala?Ser?Lys?Lys?Asp?Asn?Glu?His?His

435?????????????????440?????????????????445

Val?Phe?Lys?Val?Lys?Asp?Met?Glu?Asp?Leu?Glu?Asn?Val?Phe?Tyr?Gln

450?????????????????455?????????????????460

Met?Ile?Asp?Glu?Thr?Lys?Ser?Leu?Ser?Leu?Cys?Gly?Met?Val?Trp?Glu

465?????????????????470?????????????????475?????????????????480

His?Lys?Lys?Gly?Asn?Asp?Tyr?His?Lys?Gln?Pro?Trp?Gln?Ala?Lys?Ile

485?????????????????490?????????????????495

Ser?Val?Thr?Arg?Pro?Leu?Lys?Gly?His?Glu?Thr?Cys?Met?Gly?Ala?Val

500?????????????????505?????????????????510

Val?Ser?Glu?Tyr?Phe?Val?Leu?Thr?Ala?Ala?His?Cys?Phe?Met?Val?Asp

515?????????????????520?????????????????525

Asp?Gln?Lys?His?Ser?Ile?Lys?Val?Ser?Val?Gly?Gly?Gln?Arg?Arg?Asp

530?????????????????535?????????????????540

Leu?Glu?Ile?Glu?Glu?Val?Leu?Phe?His?Pro?Lys?Tyr?Asn?Ile?Asn?Gly

545?????????????????550?????????????????555?????????????????560

Lys?Lys?Ala?Glu?Gly?Ile?Pro?Glu?Phe?Tyr?Asp?Tyr?Asp?Val?Ala?Leu

565?????????????????570?????????????????575

Val?Lys?Leu?Lys?Asn?Lys?Leu?Lys?Tyr?Gly?Gln?Thr?Leu?Arg?Pro?Ile

580?????????????????585?????????????????590

Cys?Leu?Pro?Cys?Thr?Glu?Gly?Thr?Thr?Arg?Ala?Leu?Arg?Leu?Pro?Gln

595?????????????????600?????????????????605

Thr?Ala?Thr?Cys?Lys?Gln?His?Lys?Glu?Gln?Leu?Leu?Pro?Val?Lys?Asp

610?????????????????615?????????????????620

Val?Lys?Ala?Leu?Phe?Val?Ser?Glu?Gln?Gly?Lys?Ser?Leu?Thr?Arg?Lys

625?????????????????630?????????????????635?????????????????640

Glu?Val?Tyr?Ile?Lys?Asn?Gly?Asp?Lys?Lys?Ala?Ser?Cys?Glu?Arg?Asp

645?????????????????650?????????????????655

Ala?Thr?Lys?Ala?Gln?Gly?Tyr?Glu?Lys?Val?Lys?Asp?Ala?Ser?Glu?Val

660?????????????????665?????????????????670

Val?Thr?Pro?Arg?Phe?Leu?Cys?Thr?Gly?Gly?Val?Asp?Pro?Tyr?Ala?Asp

675?????????????????680?????????????????685

Pro?Asn?Thr?Cys?Lys?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ile?Val?His?Lys

690?????????????????695?????????????????700

Arg?Ser?Arg?Phe?Ile?Gln?Val?Gly?Val?Ile?Ser?Trp?Gly?Val?Val?Asp

705?????????????????710?????????????????715?????????????????720

Val?Cys?Arg?Asp?Gln?Arg?Arg?Gln?Gln?Leu?Val?Pro?Ser?Tyr?Ala?Arg

725?????????????????730?????????????????735

Asp?Phe?His?Ile?Asn?Leu?Phe?Gln?Val?Leu?Pro?Trp?Leu?Lys?Asp?Lys

740?????????????????745?????????????????750

Leu?Lys?Asp?Glu?Asp?Leu?Gly?Phe?Leu

755?????????????????760

<210>15

<211>2428

<212>DNA

＜213〉mouse kind (Mus sp.)

<400>15

tgtagccaga?tccagcattt?gggtttcagt?ttggacagga?ggtcaaatag?gcacccagag????60

tgacctggag?agggctttgg?gccactggac?tctctggtgc?tttccatgac?aatggagagc???120

ccccagctct?gcctcgtcct?cttggtctta?ggcttctcct?ctggaggtgt?gagcgcaact???180

ccagtgcttg?aggcccggcc?ccaagtctcc?tgctctctgg?agggagtaga?gatcaaaggc???240

ggctcctttc?aacttctcca?aggcggtcag?gccctggagt?acctatgtcc?ctctggcttc???300

tacccatacc?ccgtgcagac?tcgaacctgc?agatccacag?gctcctggag?cgacctgcag???360

acccgagacc?aaaagattgt?ccagaaggcg?gaatgcagag?caatacgctg?cccacgaccg???420

caggactttg?aaaatgggga?attctggccc?cggtccccct?tctacaacct?gagtgaccag???480

atttcttttc?aatgctatga?tggttacgtt?ctccggggct?ctgctaatcg?cacctgccaa???540

gagaatggcc?ggtgggatgg?gcaaacagca?atttgtgatg?atggagctgg?atactgtccc???600

aatcccggta?ttcctattgg?gacaaggaag?gtgggtagcc?aataccgcct?tgaagacatt???660

gttacttacc?actgcagccg?gggacttgtc?ctgcgtggct?cccagaagcg?aaagtgtcaa???720

gaaggtggct?catggagtgg?gacagagcct?tcctgccaag?attccttcat?gtatgacagc???780

cctcaagaag?tggccgaagc?attcctatcc?tccctgacag?agaccatcga?aggagccgat???840

gctgaggatg?ggcacagccc?aggagaacag?cagaagagga?agattgtcct?agacccctcg???900

ggctccatga?atatctacct?ggtgctagat?ggatcagaca?gcatcggaag?cagcaacttc???960

acaggggcta?agcggtgcct?caccaacttg?attgagaagg?tggcgagtta?cggggtgagg??1020

ccacgatatg?gtctcctgac?atatgctaca?gtccccaaag?tgttggtcag?agtgtctgat??1080

gagaggagta?gcgatgccga?ctgggtcaca?gagaagctca?accaaatcag?ttatgaagac??1140

cacaagctga?agtcagggac?caacaccaag?agggctctcc?aggctgtgta?tagcatgatg??1200

agctgggcag?gggatgcccc?gcctgaaggc?tggaatagaa?cccgccatgt?catcatcatt??1260

atgactgatg?gcttgcacaa?catgggtgga?aaccctgtca?ctgtcattca?ggacatccga??1320

gccttgctgg?acatcggcag?ggatcccaaa?aatcccaggg?aggattacct?ggatgtgtat??1380

gtgtttgggg?tcgggcctct?ggtggactcc?gtgaacatca?atgccttagc?ttccaaaaag??1440

gacaatgagc?atcatgtgtt?taaagtcaag?gatatggaag?acctggagaa?tgttttctac??1500

caaatgattg?atgaaaccaa?atctctgagt?ctctgtggca?tggtgtggga?gcataaaaaa??1560

ggcaacgatt?atcataagca?accatggcaa?gccaagatct?cagtcactcg?ccctctgaaa??1620

ggacatgaga?cctgtatggg?ggccgtggtg?tctgagtact?tcgtgctgac?agcagcgcac??1680

tgcttcatgg?tggatgatca?gaaacattcc?atcaaggtca?gcgtgggggg?tcagaggcgg??1740

gacctggaga?ttgaagaggt?cctgttccac?cccaaataca?atattaatgg?gaaaaaggca??1800

gaagggatcc?ctgagttcta?tgattatgat?gtggccctag?tcaagctcaa?gaacaagctc??1860

aagtatggcc?agactctcag?gcccatctgt?ctcccctgca?cggagggaac?cacacgagcc??1920

ttgaggcttc?ctcagacagc?cacctgcaag?cagcacaagg?aacagttgct?ccctgtgaag??1980

gatgtcaaag?ctctgtttgt?atctgagcaa?gggaagagcc?tgactcggaa?ggaggtgtac??2040

atcaagaatg?gggacaagaa?agccagttgt?gagagagatg?ctacaaaggc?ccaaggctat??2100

gagaaggtca?aagatgcctc?tgaggtggtc?actccacggt?tcctctgcac?aggaggggtg??2160

gatccctatg?ctgaccccaa?cacatgcaaa?ggagattccg?ggggccctct?cattgttcac??2220

aagagaagcc?gcttcattca?agttggtgtg?attagctggg?gagtagtaga?tgtctgcaga??2280

gaccagaggc?ggcaacagct?ggtaccctct?tatgcccggg?acttccacat?caacctcttc??2340

caggtgctgc?cctggctaaa?ggacaagctc?aaagatgagg?atttgggttt?tctataaaga??2400

gcttcctgca?gggagagtgt?gaggacag?????????????????????????????????????2428

<210>16

<211>761

<212>PRT

＜213〉mouse kind (Mus sp.)

<400>16

Met?Glu?Ser?Pro?Gln?Leu?Cys?Leu?Val?Leu?Leu?Val?Leu?Gly?Phe?Ser

1???????????????5???????????????????10??????????????????15

Ser?Gly?Gly?Val?Ser?Ala?Thr?Pro?Val?Leu?Glu?Ala?Arg?Pro?Gln?Val

20??????????????????25??????????????????30

Ser?Cys?Ser?Leu?Glu?Gly?Val?Glu?Ile?Lys?Gly?Gly?Ser?Phe?Gln?Leu

35??????????????????40??????????????????45

Leu?Gln?Gly?Gly?Gln?Ala?Leu?Glu?Tyr?Leu?Cys?Pro?Ser?Gly?Phe?Tyr

50??????????????????55??????????????????60

Pro?Tyr?Pro?Val?Gln?Thr?Arg?Thr?Cys?Arg?Ser?Thr?Gly?Ser?Trp?Ser

65??????????????????70??????????????????75??????????????????80

Asp?Leu?Gln?Thr?Arg?Asp?Gln?Lys?Ile?Val?Gln?Lys?Ala?Glu?Cys?Arg

85??????????????????90??????????????????95

Ala?Ile?Arg?Cys?Pro?Arg?Pro?Gln?Asp?Phe?Glu?Asn?Gly?Glu?Phe?Trp

100?????????????????105?????????????????110

Pro?Arg?Ser?Pro?Phe?Tyr?Asn?Leu?Ser?Asp?Gln?Ile?Ser?Phe?Gln?Cys

115?????????????????120?????????????????125

Tyr?Asp?Gly?Tyr?Val?Leu?Arg?Gly?Ser?Ala?Asn?Arg?Thr?Cys?Gln?Glu

130?????????????????135?????????????????140

Asn?Gly?Arg?Trp?Asp?Gly?Gln?Thr?Ala?Ile?Cys?Asp?Asp?Gly?Ala?Gly

145?????????????????150?????????????????155?????????????????160

Tyr?Cys?Pro?Asn?Pro?Gly?Ile?Pro?Ile?Gly?Thr?Arg?Lys?Val?Gly?Ser

165?????????????????170?????????????????175

Gln?Tyr?Arg?Leu?Glu?Asp?Ile?Val?Thr?Tyr?His?Cys?Ser?Arg?Gly?Leu

180?????????????????185?????????????????190

Val?Leu?Arg?Gly?Ser?Gln?Lys?Arg?Lys?Cys?Gln?Glu?Gly?Gly?Ser?Trp

195?????????????????200?????????????????205

Ser?Gly?Thr?Glu?Pro?Ser?Cys?Gln?Asp?Ser?Phe?Met?Tyr?Asp?Ser?Pro

210?????????????????215?????????????????220

Gln?Glu?Val?Ala?Glu?Ala?Phe?Leu?Ser?Ser?Leu?Thr?Glu?Thr?Ile?Glu

225?????????????????230?????????????????235?????????????????240

Gly?Ala?Asp?Ala?Glu?Asp?Gly?His?Ser?Pro?Gly?Glu?Gln?Gln?Lys?Arg

245?????????????????250?????????????????255

Lys?Ile?Val?Leu?Asp?Pro?Ser?Gly?Ser?Met?Asn?Ile?Tyr?Leu?Val?Leu

260?????????????????265?????????????????270

Asp?Gly?Ser?Asp?Ser?Ile?Gly?Ser?Ser?Asn?Phe?Thr?Gly?Ala?Lys?Arg

275?????????????????280?????????????????285

Cys?Leu?Thr?Asn?Leu?Ile?Glu?Lys?Val?Ala?Ser?Tyr?Gly?Val?Arg?Pro

290?????????????????295?????????????????300

Arg?Tyr?Gly?Leu?Leu?Thr?Tyr?Ala?Thr?Val?Pro?Lys?Val?Leu?Val?Arg

305?????????????????310?????????????????315?????????????????320

Val?Ser?Asp?Glu?Arg?Ser?Ser?Asp?Ala?Asp?Trp?Val?Thr?Glu?Lys?Leu

325?????????????????330?????????????????335

Asn?Gln?Ile?Ser?Tyr?Glu?Asp?His?Lys?Leu?Lys?Ser?Gly?Thr?Asn?Thr

340?????????????????345?????????????????350

Lys?Arg?Ala?Leu?Gln?Ala?Val?Tyr?Ser?Met?Met?Ser?Trp?Ala?Gly?Asp

355?????????????????360?????????????????365

Ala?Pro?Pro?Glu?Gly?Trp?Asn?Arg?Thr?Arg?His?Val?Ile?Ile?Ile?Met

370?????????????????375?????????????????380

Thr?Asp?Gly?Leu?His?Asn?Met?Gly?Gly?Asn?Pro?Val?Thr?Val?Ile?Gln

385?????????????????390?????????????????395?????????????????400

Asp?Ile?Arg?Ala?Leu?Leu?Asp?Ile?Gly?Arg?Asp?Pro?Lys?Asn?Pro?Arg

405?????????????????410?????????????????415

Glu?Asp?Tyr?Leu?Asp?Val?Tyr?Val?Phe?Gly?Val?Gly?Pro?Leu?Val?Asp

420?????????????????425?????????????????430

Ser?Val?Asn?Ile?Asn?Ala?Leu?Ala?Ser?Lys?Lys?Asp?Asn?Glu?His?His

435?????????????????440?????????????????445

Val?Phe?Lys?Val?Lys?Asp?Met?Glu?Asp?Leu?Glu?Asn?Val?Phe?Tyr?Gln

450?????????????????455?????????????????460

Met?Ile?Asp?Glu?Thr?Lys?Ser?Leu?Ser?Leu?Cys?Gly?Met?Val?Trp?Glu

465?????????????????470?????????????????475?????????????????480

His?Lys?Lys?Gly?Asn?Asp?Tyr?His?Lys?Gln?Pro?Trp?Gln?Ala?Lys?Ile

485?????????????????490?????????????????495

Ser?Val?Thr?Arg?Pro?Leu?Lys?Gly?His?Glu?Thr?Cys?Met?Gly?Ala?Val

500?????????????????505?????????????????510

Val?Ser?Glu?Tyr?Phe?Val?Leu?Thr?Ala?Ala?His?Cys?Phe?Met?Val?Asp

515?????????????????520?????????????????525

Asp?Gln?Lys?His?Ser?Ile?Lys?Val?Ser?Val?Gly?Gly?Gln?Arg?Arg?Asp

530?????????????????535?????????????????540

Leu?Glu?Ile?Glu?Glu?Val?Leu?Phe?His?Pro?Lys?Tyr?Asn?Ile?Asn?Gly

545?????????????????550?????????????????555?????????????????560

Lys?Lys?Ala?Glu?Gly?Ile?Pro?Glu?Phe?Tyr?Asp?Tyr?Asp?Val?Ala?Leu

565?????????????????570?????????????????575

Val?Lys?Leu?Lys?Asn?Lys?Leu?Lys?Tyr?Gly?Gln?Thr?Leu?Arg?Pro?Ile

580?????????????????585?????????????????590

Cys?Leu?Pro?Cys?Thr?Glu?Gly?Thr?Thr?Arg?Ala?Leu?Arg?Leu?Pro?Gln

595?????????????????600?????????????????605

Thr?Ala?Thr?Cys?Lys?Gln?His?Lys?Glu?Gln?Leu?Leu?Pro?Val?Lys?Asp

610?????????????????615?????????????????620

Val?Lys?Ala?Leu?Phe?Val?Ser?Glu?Gln?Gly?Lys?Ser?Leu?Thr?Arg?Lys

625?????????????????630?????????????????635?????????????????640

Glu?Val?Tyr?Ile?Lys?Asn?Gly?Asp?Lys?Lys?Ala?Ser?Cys?Glu?Arg?Asp

645?????????????????650?????????????????655

Ala?Thr?Lys?Ala?Gln?Gly?Tyr?Glu?Lys?Val?Lys?Asp?Ala?Ser?Glu?Val

660?????????????????665?????????????????670

Val?Thr?Pro?Arg?Phe?Leu?Cys?Thr?Gly?Gly?Val?Asp?Pro?Tyr?Ala?Asp

675?????????????????680?????????????????685

Pro?Asn?Thr?Cys?Lys?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ile?Val?His?Lys

690?????????????????695?????????????????700

Arg?Ser?Arg?Phe?Ile?Gln?Val?Gly?Val?Ile?Ser?Trp?Gly?Val?Val?Asp

705?????????????????710?????????????????715?????????????????720

Val?Cys?Arg?Asp?Gln?Arg?Arg?Gln?Gln?Leu?Val?Pro?Ser?Tyr?Ala?Arg

725?????????????????730?????????????????735

Asp?Phe?His?Ile?Asn?Leu?Phe?Gln?Val?Leu?Pro?Trp?Leu?Lys?Asp?Lys

740?????????????????745?????????????????750

Leu?Lys?Asp?Glu?Asp?Leu?Gly?Phe?Leu

755?????????????????760

<210>17

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic primer

<400>17

ctagctagct?cctgccccag?gcccagcttc?tctcc???????????????????????????????35

<210>18

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic primer

<400>18

ctagctagct?caatcccacg?cccctgtcc??????????????????????????????????????29

<210>19

<211>7912

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic polyribonucleotides

<400>19

ctaaattgta?agcgttaata?ttttgttaaa?attcgcgtta?aatttttgtt?aaatcagctc????60

attttttaac?caataggccg?aaatcggcaa?aatcccttat?aaatcaaaag?aatagaccga???120

gatagggttg?agtgttgttc?cagtttggaa?caagagtcca?ctattaaaga?acgtggactc???180

caacgtcaaa?gggcgaaaaa?ccgtctatca?gggcgatggc?ccactacgtg?aaccatcacc???240

ctaatcaagt?tttttggggt?cgaggtgccg?taaagcacta?aatcggaacc?ctaaagggag???300

cccccgattt?agagcttgac?ggggaaagcc?ggcgaacgtg?gcgagaaagg?aagggaagaa???360

agcgaaagga?gcgggcgcta?gggcgctggc?aagtgtagcg?gtcacgctgc?gcgtaaccac???420

cacacccgcc?gcgcttaatg?cgccgctaca?gggcgcgtcc?cattcgccat?tcaggctgcg???480

caactgttgg?gaagggcgat?cggtgcgggc?ctcttcgcta?ttacgccagc?tggcgaaagg???540

gggatgtgct?gcaaggcgat?taagttgggt?aacgccaggg?ttttcccagt?cacgacgttg???600

taaaacgacg?gccagtgagc?gcgcgagctc?caccgcggtg?gcggccgctc?tagaactctc???660

agtacaatct?gctctgatgc?cgcatagtta?agccagtatc?tgctccctgc?ttgtgtgttg???720

gaggtcgctg?agtagtgcgc?gagcaaaatt?taagctacaa?caaggcaagg?cttgaccgac???780

aattgcatga?agaatctgct?tagggttagg?cgttttgcgc?tgcttcgcga?tgtacgggcc???840

agatatacgc?gtatctgagg?ggactagggt?gtgtttaggc?gaaaagcggg?gcttcggttg???900

tacgcggtta?ggagtcccct?caggatatag?tagtttcgct?tttgcatagg?gagggggaaa???960

tgtagtctta?tgcaatactc?ttgtagtctt?gcaacatggt?aacgatgagt?tagcaacatg??1020

ccttacaagg?agagaaaaag?caccgtgcat?gccgattggt?ggaagtaagg?tggtacgatc??1080

gtgccttatt?aggaaggcaa?cagacgggtc?tgacatggat?tggacgaacc?actgaattcc??1140

gcattgcaga?gatattgtat?ttaagtgcct?agctcgatac?aataaacgcc?atttgaccat??1200

tcaccacatt?ggtgtgcacc?tccaatctag?aactagtgta?agtatcaagg?ttacaagaca??1260

ggtttaagga?gaccaataga?aactgggctt?gtcgagacag?agaagactct?tgcgtttctg??1320

ataggcacct?attggtctta?ctgacatcca?ctttgccttt?ctctccacag?actagtggat??1380

ctcgagatga?agcggagaga?gctggagaag?aaactgagga?aagtgcgcgt?gacacctcaa??1440

caggacaagt?actataccat?cggcaacctg?cagtgggcca?tccgcatgat?caacctgatg??1500

ggcatcaagt?gcgtgtgcga?cgaggaatgc?agcgccgctg?aggtcgccct?gatcatcacc??1560

cagtttagcg?ccctcgacct?ggagaactcc?cctatccgcg?gcaaggaaga?ggtggccatc??1620

aagaataccc?tgaaggtgtt?ttggagcctg?ctggccggat?acaagcctga?gagcaccgag??1680

accgccctgg?gatactggga?agccttcacc?tacagagaga?gggaagctag?agccgacaag??1740

gagggagaga?tcaagagcat?ctaccctagc?ctgacccaga?acacccagaa?caagaaacag??1800

accagcaatc?agacaaacac?ccagagcctg?cccgctatca?ccacacagga?tggcacccct??1860

cgcttcgacc?ccgacctgat?gaagcagctg?aagatctggt?ccgatgccac?agagcgcaat??1920

ggagtggacc?tgcatgccgt?gaacatcctg?ggagtgatca?cagccaacct?ggtgcaagaa??1980

gagatcaagc?tcctgctgaa?tagcacaccc?aagtggcgcc?tggacgtgca?gctgatcgag??2040

agcaaagtga?gagagaagga?gaacgcccac?cgcacctgga?agcagcatca?ccctgaggct??2100

cccaagacag?acgagatcat?tggaaaggga?ctgagctccg?ccgagcaggc?taccctgatc??2160

agcgtggagt?gcagagagac?cttccgccag?tgggtgctgc?aggctgccat?ggaggtcgcc??2220

caggctaagc?acgccacacc?cggacctatc?aacatccatc?aaggccctaa?ggaaccctac??2280

accgacttca?tcaaccgcct?ggtggctgcc?ctggaaggaa?tggccgctcc?cgagaccaca??2340

aaggagtacc?tcctgcagca?cctgagcatc?gaccacgcca?acgaggactg?tcagtccatc??2400

ctgcgccctc?tgggacccaa?cacacctatg?gagaagaaac?tggaggcctg?tcgcgtggtg??2460

ggaagccaga?agagcaagat?gcagttcctg?gtggccgcta?tgaaggaaat?ggggatccag??2520

tctcctattc?cagccgtgct?gcctcacaca?cccgaagcct?acgcctccca?aacctcaggg??2580

cccgaggatg?gtaggagatg?ttacggatgt?gggaagacag?gacatttgaa?gaggaattgt??2640

aaacagcaaa?aatgctacca?ttgtggcaaa?cctggccacc?aagcaagaaa?ctgcaggtca??2700

aaaaacggga?agtgctcctc?tgccccttat?gggcagagga?gccaaccaca?gaacaatttt??2760

caccagagca?acatgagttc?tgtgacccca?tctgcacccc?ctcttatatt?agattagaca??2820

aacagccttt?tataaaggtg?ttcattggcg?gccgctgggt?gaagggactg?gtggactcgg??2880

gcgctgacga?ggtggtgctg?aagaacatcc?actgggaccg?catcaaaggc?taccctggaa??2940

cacccatcaa?gcagatcggc?gtgaacggcg?tgaacgtggc?taagcgcaaa?acacatgtgg??3000

agtggagatt?caaagacaag?accggcatca?ttgacgtcct?cttcagcgac?acacctgtga??3060

acctgtttgg?cagaagcctg?ctcagatcca?tcgtgacctg?ctttaccctg?ctggtgcaca??3120

ccgagaagat?cgagccactg?cctgtgaagg?tgcgcggccc?tggacctaag?gtgccacaat??3180

ggcccctgac?caaggagaaa?taccaggccc?tgaaggagat?cgtgaaggac?ctgctggccg??3240

agggaaagat?cagcgaagct?gcctgggaca?acccttacaa?cacacccgtg?ttcgtgatca??3300

agaagaaagg?caccggccgc?tggcgcatgc?tgatggactt?ccgcgagctg?aataagatca??3360

ccgtgaaagg?ccaagagttc?agcacaggac?tcccttatcc?acccggcatc?aaggagtgtg??3420

agcacctgac?cgccatcgac?atcaaggacg?cctacttcac?catccctctg?cacgaggact??3480

tcagaccctt?cacagccttc?agcgtggtcc?cagtgaaccg?cgagggcccc?atcgagcgct??3540

tccagtggaa?cgtcctgcct?caaggctggg?tgtgctcccc?tgccatctac?cagaccacaa??3600

cccagaagat?cattgagaac?atcaagaaga?gccatcccga?cgtgatgctg?tatcagtaca??3660

tggatgacct?cctgattggc?agcaatcgcg?atgaccacaa?gcagatcgtg?caggagatca??3720

gagacaagct?gggcagctat?ggcttcaaga?cacccgacga?gaaagtgcag?gaagagcgcg??3780

tgaagtggat?cggcttcgag?ctgacaccta?agaaatggag?attccagcct?aggcaactga??3840

agatcaagaa?cccactgacc?gtgaacgaac?tccagcagct?ggtcggcaac?tgtgtgtggg??3900

tgcagcccga?ggtgaagatc?cctctgtacc?cactgaccga?tctgctccgc?gacaagacca??3960

acctgcagga?aaagatccag?ctgacacccg?aggccatcaa?gtgcgtggaa?gagttcaacc??4020

tgaagctgaa?agatcccgag?tggaaggaca?gaattcgcga?aggagccgag?ctggtgatca??4080

agatccaaat?ggtccctcgc?ggcatcgtgt?tcgacctgct?gcaagacggc?aatcctatct??4140

ggggaggcgt?gaaaggactg?aactacgacc?acagcaacaa?gatcaagaag?atcctgcgca??4200

ccatgaacga?gctgaaccgc?accgtggtga?tcatgaccgg?acgcgaagct?agctttctcc??4260

tgcctggatc?cagcgaggat?tgggaggccg?ccctgcagaa?ggaagagagc?ctgacccaaa??4320

tctttcccgt?gaagttctac?cgccatagct?gtagatggac?aagcatctgt?ggacccgtcc??4380

gcgagaacct?gaccacctac?tataccgacg?gcgggaagaa?aggaaagaca?gctgccgcag??4440

tgtactggtg?tgaaggaaga?actaagagca?aagtgttccc?tggaaccaat?caacaggctg??4500

agctgaaggc?aatctgcatg?gctctgctgg?acggacctcc?caagatgaac?atcatcaccg??4560

acagccgcta?cgcttatgag?ggcatgagag?aggaacctga?gacctgggct?cgcgagggca??4620

tctggctgga?gattgcaaag?atcctgccat?tcaagcaata?cgtcggagtg?ggctgggtcc??4680

ctgctcacaa?aggcattgga?ggcaataccg?aggctgacga?aggagtgaag?aaagccctgg??4740

agcaaatggc?accatgttcc?cctcccgagg?ctatcctgct?caaacctggc?gagaagcaaa??4800

acctggagac?cggcatctac?atgcaaggcc?tgagacctca?gagcttcctg?ccccgcgctg??4860

acctccctgt?cgcaatcact?ggcaccatgg?tggactccga?gctgcagctc?caactgctga??4920

acatcggcac?cgagcacatt?cgcatccaga?aggacgaggt?gttcatgaca?tgcttcctgg??4980

agaacatccc?tagcgccacc?gaagaccacg?agagatggca?cacatcccca?gacatcctgg??5040

tccgccagtt?ccacctgccc?aagcgcatcg?ccaaggagat?cgtcgcccgc?tgccaggagt??5100

gcaagagaac?cacaacctcc?ccagtgcgcg?gcaccaaccc?tagaggacgc?ttcctgtggc??5160

agatggacaa?cacacactgg?aacaaaacca?tcatttgggt?cgcagtggag?actaacagcg??5220

gactggtgga?ggctcaggtg?attcccgaag?agaccgcact?gcaagtggcc?ctgtgtatcc??5280

tccagctgat?ccaacgctac?accgtcctgc?acctgcacag?cgacaacgga?ccctgcttca??5340

cagctcaccg?catcgagaac?ctgtgcaagt?acctgggcat?caccaagaca?accggcattc??5400

cctacaatcc?tcagagccaa?ggagtcgtgg?aaagagccca?tcgcgacctg?aaggacagac??5460

tggctgccta?tcaaggcgac?tgcgagaccg?tggaagctgc?actgagcctc?gccctggtca??5520

gcctgaacaa?gaagagagga?ggcatcggcg?gacacacacc?ctacgagatc?tatctggaga??5580

gcgagcacac?caagtatcag?gaccaactgg?agcagcaatt?cagcaagcag?aagatcgaga??5640

aatggtgcta?cgtccgcaac?agacgcaagg?agtggaaggg?cccttacaag?gtgctgtggg??5700

atggcgacgg?agctgcagtg?atcgaggaag?agggcaagac?cgctctgtat?ccccaccggc??5760

acatgcgctt?catcccacct?cccgacagcg?atatccagga?cggctccagc?tgatctagag??5820

tcgcccttta?agctgcaata?aacaatcatt?attttcattg?gatctgtgtg?ttggtttttt??5880

gtgtgggctt?gggggagggg?gaggccagaa?tgactccaag?agctacagga?aggcaggtca??5940

gagaccccac?tggacaaaca?gtggctggac?tctgcaccat?aacacacaat?caacagggga??6000

gtgagctgga?tcgagctggg?gggatcctct?aggatccccc?gggctgcagg?aattcgatat??6060

caagcttatc?gataccgtcg?acctcgaggg?ggggcccggt?accacatgtg?agcaaaaggc??6120

cagcaaaagg?ccaggaaccg?taaaaaggcc?gcgttgctgg?cgtttttcca?taggctccgc??6180

ccccctgacg?agcatcacaa?aaatcgacgc?tcaagtcaga?ggtggcgaaa?cccgacagga??6240

ctataaagat?accaggcgtt?tccccctgga?agctccctcg?tgcgctctcc?tgttccgacc??6300

ctgccgctta?ccggatacct?gtccgccttt?ctcccttcgg?gaagcgtggc?gctttctcat??6360

agctcacgct?gtaggtatct?cagttcggtg?taggtcgttc?gctccaagct?gggctgtgtg??6420

cacgaacccc?ccgttcagcc?cgaccgctgc?gccttatccg?gtaactatcg?tcttgagtcc??6480

aacccggtaa?gacacgactt?atcgccactg?gcagcagcca?ctggtaacag?gattagcaga??6540

gcgaggtatg?taggcggtgc?tacagagttc?ttgaagtggt?ggcctaacta?cggctacact??6600

agaagaacag?tatttggtat?ctgcgctctg?ctgaagccag?ttaccttcgg?aaaaagagtt??6660

ggtagctctt?gatccggcaa?acaaaccacc?gctggtagcg?gtggtttttt?tgtttgcaag??6720

cagcagatta?cgcgcagaaa?aaaaggatct?caagaagatc?ctttgatctt?ttctacgggg??6780

tctgacgctc?agtggaacga?aaactcacgt?taagggattt?tggtcatgag?attatcaaaa??6840

aggatcttca?cctagatcct?tttaaattaa?aaatgaagtt?ttaaatcaat?ctaaagtata??6900

tatgagtaaa?cttggtctga?cagttaccaa?tgcttaatca?gtgaggcacc?tatctcagcg??6960

atctgtctat?ttcgttcatc?catagttgcc?tgactccccg?tcgtgtagat?aactacgata??7020

cgggagggct?taccatctgg?ccccagtgct?gcaatgatac?cgcgagaccc?acgctcaccg??7080

gctccagatt?tatcagcaat?aaaccagcca?gccggaaggg?ccgagcgcag?aagtggtcct??7140

gcaactttat?ccgcctccat?ccagtctatt?aattgttgcc?gggaagctag?agtaagtagt??7200

tcgccagtta?atagtttgcg?caacgttgtt?gccattgcta?caggcatcgt?ggtgtcacgc??7260

tcgtcgtttg?gtatggcttc?attcagctcc?ggttcccaac?gatcaaggcg?agttacatga??7320

tcccccatgt?tgtgcaaaaa?agcggttagc?tccttcggtc?ctccgatcgt?tgtcagaagt??7380

aagttggccg?cagtgttatc?actcatggtt?atggcagcac?tgcataattc?tcttactgtc??7440

atgccatccg?taagatgctt?ttctgtgact?ggtgagtact?caaccaagtc?attctgagaa??7500

tagtgtatgc?ggcgaccgag?ttgctcttgc?ccggcgtcaa?tacgggataa?taccgcgcca??7560

catagcagaa?ctttaaaagt?gctcatcatt?ggaaaacgtt?cttcggggcg?aaaactctca??7620

aggatcttac?cgctgttgag?atccagttcg?atgtaaccca?ctcgtgcacc?caactgatct??7680

tcagcatctt?ttactttcac?cagcgtttct?gggtgagcaa?aaacaggaag?gcaaaatgcc??7740

gcaaaaaagg?gaataagggc?gacacggaaa?tgttgaatac?tcatactctt?cctttttcaa??7800

tattattgaa?gcatttatca?gggttattgt?ctcatgagcg?gatacatatt?tgaatgtatt??7860

tagaaaaata?aacaaatagg?ggttccgcgc?acatttcccc?gaaaagtgcc?ac??????????7912

<210>20

<211>4026

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic polyribonucleotides

<400>20

ctaaattgta?agcgttaata?ttttgttaaa?attcgcgtta?aatttttgtt?aaatcagctc????60

attttttaac?caataggccg?aaatcggcaa?aatcccttat?aaatcaaaag?aatagaccga???120

gatagggttg?agtgttgttc?cagtttggaa?caagagtcca?ctattaaaga?acgtggactc???180

caacgtcaaa?gggcgaaaaa?ccgtctatca?gggcgatggc?ccactacgtg?aaccatcacc???240

ctaatcaagt?tttttggggt?cgaggtgccg?taaagcacta?aatcggaacc?ctaaagggag???300

cccccgattt?agagcttgac?ggggaaagcc?ggcgaacgtg?gcgagaaagg?aagggaagaa???360

agcgaaagga?gcgggcgcta?gggcgctggc?aagtgtagcg?gtcacgctgc?gcgtaaccac???420

cacacccgcc?gcgcttaatg?cgccgctaca?gggcgcgtcc?cattcgccat?tcaggctgcg???480

caactgttgg?gaagggcgat?cggtgcgggc?ctcttcgcta?ttacgccagc?tggcgaaagg???540

gggatgtgct?gcaaggcgat?taagttgggt?aacgccaggg?ttttcccagt?cacgacgttg???600

taaaacgacg?gccagtgagc?gcgcgagctc?caccgcggtg?gcggccgctc?tagaactctc???660

agtacaatct?gctctgatgc?cgcatagtta?agccagtatc?tgctccctgc?ttgtgtgttg???720

gaggtcgctg?agtagtgcgc?gagcaaaatt?taagctacaa?caaggcaagg?cttgaccgac???780

aattgcatga?agaatctgct?tagggttagg?cgttttgcgc?tgcttcgcga?tgtacgggcc???840

agatatacgc?gtatctgagg?ggactagggt?gtgtttaggc?gaaaagcggg?gcttcggttg???900

tacgcggtta?ggagtcccct?caggatatag?tagtttcgct?tttgcatagg?gagggggaaa???960

tgtagtctta?tgcaatactc?ttgtagtctt?gcaacatggt?aacgatgagt?tagcaacatg??1020

ccttacaagg?agagaaaaag?caccgtgcat?gccgattggt?ggaagtaagg?tggtacgatc??1080

gtgccttatt?aggaaggcaa?cagacgggtc?tgacatggat?tggacgaacc?actgaattcc??1140

gcattgcaga?gatattgtat?ttaagtgcct?agctcgatac?aataaacgcc?atttgaccat??1200

tcaccacatt?ggtgtgcacc?tccaatctag?aactagtgta?agtatcaagg?ttacaagaca??1260

ggtttaagga?gaccaataga?aactgggctt?gtcgagacag?agaagactct?tgcgtttctg??1320

ataggcacct?attggtctta?ctgacatcca?ctttgccttt?ctctccacag?actagtggat??1380

cgatccgcca?ccatggacca?ggacctggac?agagctgaga?gaggcgagag?aggcggcgga??1440

agcgaggaac?tgctgcagga?agagatcaac?gagggacggc?tgacagctag?agaggccctg??1500

cagacctgga?tcaacaatga?tagccctcgg?tacgtgaaga?aactgagaca?gggacagccc??1560

gaactgccca?ccagccctgg?aggcggaggc?ggaagaggac?acagagctag?gaagctgcct??1620

ggcgagcgga?gacctggatt?ctggaagagc?ctgcgggagc?tggtggagca?gaacagacgg??1680

aagcaggaga?gacggctgag?cggactggac?agacggattc?agcagctgga?ggacctggtg??1740

agacacatga?gcctgggcag?ccctgatcct?agcacaccta?gcgccagcgt?cctgtccgtg??1800

aatcctcccg?ctcagacacc?tctgggacac?ctgccaccta?gaagctactt?caagctgaaa??1860

cgggtggact?gtggcgctgg?ctgggacctg?agaaccacag?ctgctcccgg?cctgcctatc??1920

tgcgagctgg?actggattca?gggcaccaag?tgaggatcga?tcccccgggc?tgcaggaatt??1980

cgatatcaag?cttatcgata?ccgtcgacct?cgagaataaa?caatcattat?tttcattgga??2040

tctgtgtgtt?ggttttttgt?gtgggcttgg?gggaggggga?ggccagaatg?actccaagag??2100

ctacaggaag?gcaggtcaga?gaccccactg?gacaaacagt?ggctggactc?tgcaccataa??2160

cacacaatca?acaggggagt?gagctggatc?gagctgctcg?agggggggcc?cggtaccaca??2220

tgtgagcaaa?aggccagcaa?aaggccagga?accgtaaaaa?ggccgcgttg?ctggcgtttt??2280

tccataggct?ccgcccccct?gacgagcatc?acaaaaatcg?acgctcaagt?cagaggtggc??2340

gaaacccgac?aggactataa?agataccagg?cgtttccccc?tggaagctcc?ctcgtgcgct??2400

ctcctgttcc?gaccctgccg?cttaccggat?acctgtccgc?ctttctccct?tcgggaagcg??2460

tggcgctttc?tcatagctca?cgctgtaggt?atctcagttc?ggtgtaggtc?gttcgctcca??2520

agctgggctg?tgtgcacgaa?ccccccgttc?agcccgaccg?ctgcgcctta?tccggtaact??2580

atcgtcttga?gtccaacccg?gtaagacacg?acttatcgcc?actggcagca?gccactggta??2640

acaggattag?cagagcgagg?tatgtaggcg?gtgctacaga?gttcttgaag?tggtggccta??2700

actacggcta?cactagaaga?acagtatttg?gtatctgcgc?tctgctgaag?ccagttacct??2760

tcggaaaaag?agttggtagc?tcttgatccg?gcaaacaaac?caccgctggt?agcggtggtt??2820

tttttgtttg?caagcagcag?attacgcgca?gaaaaaaagg?atctcaagaa?gatcctttga??2880

tcttttctac?ggggtctgac?gctcagtgga?acgaaaactc?acgttaaggg?attttggtca??2940

tgagattatc?aaaaaggatc?ttcacctaga?tccttttaaa?ttaaaaatga?agttttaaat??3000

caatctaaag?tatatatgag?taaacttggt?ctgacagtta?ccaatgctta?atcagtgagg??3060

cacctatctc?agcgatctgt?ctatttcgtt?catccatagt?tgcctgactc?cccgtcgtgt??3120

agataactac?gatacgggag?ggcttaccat?ctggccccag?tgctgcaatg?ataccgcgag??3180

acccacgctc?accggctcca?gatttatcag?caataaacca?gccagccgga?agggccgagc??3240

gcagaagtgg?tcctgcaact?ttatccgcct?ccatccagtc?tattaattgt?tgccgggaag??3300

ctagagtaag?tagttcgcca?gttaatagtt?tgcgcaacgt?tgttgccatt?gctacaggca??3360

tcgtggtgtc?acgctcgtcg?tttggtatgg?cttcattcag?ctccggttcc?caacgatcaa??3420

ggcgagttac?atgatccccc?atgttgtgca?aaaaagcggt?tagctccttc?ggtcctccga??3480

tcgttgtcag?aagtaagttg?gccgcagtgt?tatcactcat?ggttatggca?gcactgcata??3540

attctcttac?tgtcatgcca?tccgtaagat?gcttttctgt?gactggtgag?tactcaacca??3600

agtcattctg?agaatagtgt?atgcggcgac?cgagttgctc?ttgcccggcg?tcaatacggg??3660

ataataccgc?gccacatagc?agaactttaa?aagtgctcat?cattggaaaa?cgttcttcgg??3720

ggcgaaaact?ctcaaggatc?ttaccgctgt?tgagatccag?ttcgatgtaa?cccactcgtg??3780

cacccaactg?atcttcagca?tcttttactt?tcaccagcgt?ttctgggtga?gcaaaaacag??3840

gaaggcaaaa?tgccgcaaaa?aagggaataa?gggcgacacg?gaaatgttga?atactcatac??3900

tcttcctttt?tcaatattat?tgaagcattt?atcagggtta?ttgtctcatg?agcggataca??3960

tatttgaatg?tatttagaaa?aataaacaaa?taggggttcc?gcgcacattt?ccccgaaaag??4020

tgccac?????????????????????????????????????????????????????????????4026

<210>21

<211>5007

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic polyribonucleotides

<400>21

ctaaattgta?agcgttaata?ttttgttaaa?attcgcgtta?aatttttgtt?aaatcagctc????60

attttttaac?caataggccg?aaatcggcaa?aatcccttat?aaatcaaaag?aatagaccga???120

gatagggttg?agtgttgttc?cagtttggaa?caagagtcca?ctattaaaga?acgtggactc???180

caacgtcaaa?gggcgaaaaa?ccgtctatca?gggcgatggc?ccactacgtg?aaccatcacc???240

ctaatcaagt?tttttggggt?cgaggtgccg?taaagcacta?aatcggaacc?ctaaagggag???300

cccccgattt?agagcttgac?ggggaaagcc?ggcgaacgtg?gcgagaaagg?aagggaagaa???360

agcgaaagga?gcgggcgcta?gggcgctggc?aagtgtagcg?gtcacgctgc?gcgtaaccac???420

cacacccgcc?gcgcttaatg?cgccgctaca?gggcgcgtcc?cattcgccat?tcaggctgcg???480

caactgttgg?gaagggcgat?cggtgcgggc?ctcttcgcta?ttacgccagc?tggcgaaagg???540

gggatgtgct?gcaaggcgat?taagttgggt?aacgccaggg?ttttcccagt?cacgacgttg???600

taaaacgacg?gccagtgagc?gcgcgagctc?caccgcggtg?gcggccgctc?tagaactctc???660

agtacaatct?gctctgatgc?cgcatagtta?agccagtatc?tgctccctgc?ttgtgtgttg???720

gaggtcgctg?agtagtgcgc?gagcaaaatt?taagctacaa?caaggcaagg?cttgaccgac???780

aattgcatga?agaatctgct?tagggttagg?cgttttgcgc?tgcttcgcga?tgtacgggcc???840

agatatacgc?gtatctgagg?ggactagggt?gtgtttaggc?gaaaagcggg?gcttcggttg???900

tacgcggtta?ggagtcccct?caggatatag?tagtttcgct?tttgcatagg?gagggggaaa???960

tgtagtctta?tgcaatactc?ttgtagtctt?gcaacatggt?aacgatgagt?tagcaacatg??1020

ccttacaagg?agagaaaaag?caccgtgcat?gccgattggt?ggaagtaagg?tggtacgatc??1080

gtgccttatt?aggaaggcaa?cagacgggtc?tgacatggat?tggacgaacc?actgaattcc??1140

gcattgcaga?gatattgtat?ttaagtgcct?agctcgatac?aataaacgcc?atttgaccat??1200

tcaccacatt?ggtgtgcacc?tccaatctag?aactagtgta?agtatcaagg?ttacaagaca??1260

ggtttaagga?gaccaataga?aactgggctt?gtcgagacag?agaagactct?tgcgtttctg??1320

ataggcacct?attggtctta?ctgacatcca?ctttgccttt?ctctccacag?actagtggat??1380

cagcttgcca?ccatggtgtc?cgccatcgtg?ctgtacgtgc?tgctggccgc?tgccgcacac??1440

agcgccttcg?ccgccgagca?ctgcaacgcc?cagatgaaaa?ccggccccta?caagatcaag??1500

aacctggaca?tcaccccccc?caaggagacc?ctgcagaagg?acgtggagat?caccatcgtg??1560

gagaccgact?acaacgagaa?cgtgatcatc?ggctacaagg?gctactacca?ggcctacgcc??1620

tacaacggcg?gcagcctgga?ccccaacacc?agagtggagg?agaccatgaa?aaccctgaat??1680

gtgggcaagg?aagatctgct?gatgtggagc?atcaggcagc?agtgcgaagt?gggcgaggag??1740

ctgatcgaca?gatggggcag?cgacagcgac?gactgcttca?gagacaacga?gggcagaggc??1800

cagtgggtga?agggcaagga?gctggtgaag?cggcagaaca?acaaccactt?cgcccaccac??1860

acctgcaaca?agagctggag?atgcggcatc?agcaccagca?agatgtacag?ccggctggag??1920

tgccaggatg?acaccgatga?gtgccaggtg?tacatcctgg?acgccgaggg?caaccctatc??1980

aacgtgaccg?tggacaccgt?gctgcacaga?gatggcgtgt?ccatgatcct?gaagcagaag??2040

agcaccttca?ccaccaggca?gatcaaggcc?gcctgcctgc?tgatcaagga?cgacaagaac??2100

aaccccgaga?gcgtgaccag?agagcactgc?ctgatcgaca?acgacatcta?cgacctgagc??2160

aagaacacct?ggaactgcaa?gttcaaccgg?tgcatcaagc?ggaaagtgga?gcacagagtg??2220

aagaagagac?cccccacctg?gaggcacaat?gtgcgggcca?agtacaccga?gggcgatacc??2280

gccaccaagg?gcgacctgat?gcacatccag?gaagaactga?tgtacgagaa?cgacctgctg??2340

aagatgaaca?tcgagctgat?gcacgcccac?atcaacaagc?tgaacaacat?gctgcacgac??2400

ctgatcgtgt?ccgtggccaa?agtggacgag?aggctgatcg?gcaacctgat?gaacaacagc??2460

gtgtccagca?ccttcctgag?cgacgacacc?ttcctgctga?tgccctgcac?caacccccct??2520

gcccacacca?gcaactgcta?caacaacagc?atctacaagg?agggccggtg?ggtggccaac??2580

accgacagca?gccagtgcat?cgacttctcc?aactataagg?agctggccat?cgacgacgat??2640

gtggagttct?ggatccccac?catcggcaac?accacctacc?acgacagctg?gaaggacgcc??2700

agcggctgga?gcttcatcgc?ccagcagaag?tccaacctga?tcaccaccat?ggagaacacc??2760

aagttcggcg?gagtgggcac?cagcctgagc?gatatcacca?gcatggccga?gggcgagctg??2820

gccgccaagc?tgaccagctt?catgttcggc?cacgtggtga?acttcgtgat?catcctgatc??2880

gtgatcctgt?tcctgtactg?catgatccgg?aaccggaacc?ggcagtactg?atagtctagg??2940

atcccccggg?ctgcaggaat?tcgatatcaa?gcttatcgat?accgtcgacc?tcgagaataa??3000

acaatcatta?ttttcattgg?atctgtgtgt?tggttttttg?tgtgggcttg?ggggaggggg??3060

aggccagaat?gactccaaga?gctacaggaa?ggcaggtcag?agaccccact?ggacaaacag??3120

tggctggact?ctgcaccata?acacacaatc?aacaggggag?tgagctggat?cgagctgctc??3180

gagggggggc?ccggtaccac?atgtgagcaa?aaggccagca?aaaggccagg?aaccgtaaaa??3240

aggccgcgtt?gctggcgttt?ttccataggc?tccgcccccc?tgacgagcat?cacaaaaatc??3300

gacgctcaag?tcagaggtgg?cgaaacccga?caggactata?aagataccag?gcgtttcccc??3360

ctggaagctc?cctcgtgcgc?tctcctgttc?cgaccctgcc?gcttaccgga?tacctgtccg??3420

cctttctccc?ttcgggaagc?gtggcgcttt?ctcatagctc?acgctgtagg?tatctcagtt??3480

cggtgtaggt?cgttcgctcc?aagctgggct?gtgtgcacga?accccccgtt?cagcccgacc??3540

gctgcgcctt?atccggtaac?tatcgtcttg?agtccaaccc?ggtaagacac?gacttatcgc??3600

cactggcagc?agccactggt?aacaggatta?gcagagcgag?gtatgtaggc?ggtgctacag??3660

agttcttgaa?gtggtggcct?aactacggct?acactagaag?aacagtattt?ggtatctgcg??3720

ctctgctgaa?gccagttacc?ttcggaaaaa?gagttggtag?ctcttgatcc?ggcaaacaaa??3780

ccaccgctgg?tagcggtggt?ttttttgttt?gcaagcagca?gattacgcgc?agaaaaaaag??3840

gatctcaaga?agatcctttg?atcttttcta?cggggtctga?cgctcagtgg?aacgaaaact??3900

cacgttaagg?gattttggtc?atgagattat?caaaaaggat?cttcacctag?atccttttaa??3960

attaaaaatg?aagttttaaa?tcaatctaaa?gtatatatga?gtaaacttgg?tctgacagtt??4020

accaatgctt?aatcagtgag?gcacctatct?cagcgatctg?tctatttcgt?tcatccatag??4080

ttgcctgact?ccccgtcgtg?tagataacta?cgatacggga?gggcttacca?tctggcccca??4140

gtgctgcaat?gataccgcga?gacccacgct?caccggctcc?agatttatca?gcaataaacc??4200

agccagccgg?aagggccgag?cgcagaagtg?gtcctgcaac?tttatccgcc?tccatccagt??4260

ctattaattg?ttgccgggaa?gctagagtaa?gtagttcgcc?agttaatagt?ttgcgcaacg??4320

ttgttgccat?tgctacaggc?atcgtggtgt?cacgctcgtc?gtttggtatg?gcttcattca??4380

gctccggttc?ccaacgatca?aggcgagtta?catgatcccc?catgttgtgc?aaaaaagcgg??4440

ttagctcctt?cggtcctccg?atcgttgtca?gaagtaagtt?ggccgcagtg?ttatcactca??4500

tggttatggc?agcactgcat?aattctctta?ctgtcatgcc?atccgtaaga?tgcttttctg??4560

tgactggtga?gtactcaacc?aagtcattct?gagaatagtg?tatgcggcga?ccgagttgct??4620

cttgcccggc?gtcaatacgg?gataataccg?cgccacatag?cagaacttta?aaagtgctca??4680

tcattggaaa?acgttcttcg?gggcgaaaac?tctcaaggat?cttaccgctg?ttgagatcca??4740

gttcgatgta?acccactcgt?gcacccaact?gatcttcagc?atcttttact?ttcaccagcg??4800

tttctgggtg?agcaaaaaca?ggaaggcaaa?atgccgcaaa?aaagggaata?agggcgacac??4860

ggaaatgttg?aatactcata?ctcttccttt?ttcaatatta?ttgaagcatt?tatcagggtt??4920

attgtctcat?gagcggatac?atatttgaat?gtatttagaa?aaataaacaa?ataggggttc??4980

cgcgcacatt?tccccgaaaa?gtgccac??????????????????????????????????????5007

<210>22

<211>6329

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic polyribonucleotides

<400>22

ctaaattgta?agcgttaata?ttttgttaaa?attcgcgtta?aatttttgtt?aaatcagctc????60

attttttaac?caataggccg?aaatcggcaa?aatcccttat?aaatcaaaag?aatagaccga???120

gatagggttg?agtgttgttc?cagtttggaa?caagagtcca?ctattaaaga?acgtggactc???180

caacgtcaaa?gggcgaaaaa?ccgtctatca?gggcgatggc?ccactacgtg?aaccatcacc???240

ctaatcaagt?tttttggggt?cgaggtgccg?taaagcacta?aatcggaacc?ctaaagggag???300

cccccgattt?agagcttgac?ggggaaagcc?ggcgaacgtg?gcgagaaagg?aagggaagaa???360

agcgaaagga?gcgggcgcta?gggcgctggc?aagtgtagcg?gtcacgctgc?gcgtaaccac???420

cacacccgcc?gcgcttaatg?cgccgctaca?gggcgcgtcc?cattcgccat?tcaggctgcg???480

caactgttgg?gaagggcgat?cggtgcgggc?ctcttcgcta?ttacgccagc?tggcgaaagg???540

gggatgtgct?gcaaggcgat?taagttgggt?aacgccaggg?ttttcccagt?cacgacgttg???600

taaaacgacg?gccagtgagc?gcgcgtaata?cgactcacta?tagggcgaat?tggagctcca???660

ccgcggtggc?ggccgctcta?gaactagtgg?atcccccggg?atcccgtagt?tattaatagt???720

aatcaattac?ggggtcatta?gttcatagcc?catatatgga?gttccgcgtt?acataactta???780

cggtaaatgg?cccgcctggc?tgaccgccca?acgacccccg?cccattgacg?tcaataatga???840

cgtatgttcc?catagtaacg?ccaataggga?ctttccattg?acgtcaatgg?gtggagtatt???900

tacggtaaac?tgcccacttg?gcagtacatc?aagtgtatca?tatgccaagt?ccgcccccta???960

ttgacgtcaa?tgacggtaaa?tggcccgcct?ggcattatgc?ccagtacatg?accttacggg??1020

actttcctac?ttggcagtac?atctacgtat?tagtcatcgc?tattaccatg?gtgatgcggt??1080

tttggcagta?caccaatggg?cgtggatagc?ggtttgactc?acggggattt?ccaagtctcc??1140

accccattga?cgtcaatggg?agtttgtttt?ggcaccaaaa?tcaacgggac?tttccaaaat??1200

gtcgtaataa?ccccgccccg?ttgacgcaaa?tgggcggtag?gcgtgtacgg?tgggaggtct??1260

atataaacca?tatcttcact?ctgtacttca?gctcgtgtag?ctcattagct?ccgagctccc??1320

caacctacag?cctgagaggc?actggctcgg?ttgggtagcc?agcctttcgg?gtaataaagg??1380

cttgttggca?ttcggcatct?acccgtgcct?cctgtcttgt?cttactcgag?cgaacccaca??1440

actccgtcct?gctgagctca?cagctcgcgg?ggcggtgaag?aacacccaac?agttggcgcc??1500

caacgtgggg?ctgagtaaga?gagactcggc?tcgagtaaaa?gaagacccag?ctcgaacgag??1560

aagactccgg?acaggtgagt?agttgcgtgt?tttccccggg?gtgaagagaa?gggagttaga??1620

aaagaagctt?cgtaaggtta?gggtgacacc?ccaacaggat?aaatattata?ctatagggaa??1680

tcttcaatgg?gccattagaa?tcacgtgatg?catcgataaa?taaaaaaaga?gggggaatag??1740

ggggccatac?accatatgaa?atatacctag?aatcagaaca?taccaaatac?caagaccaac??1800

tagaacaaca?attttcaaaa?caaaaaattg?aaaagtggtg?ttacgtaagg?aacagaagaa??1860

aggaatggaa?aggaccctac?aaagtgttgt?gggacggaga?cggggcagca?gtaatagagg??1920

agaattcgtg?gattcttgta?aaggtcccca?gctatgggtt?tgtggtagta?aatgacacag??1980

atacaccacc?atccctccgc?atccgaaagc?ctcgagcagt?cggactagca?atattcctgc??2040

ttgtgctggc?tatcatggcc?atcacatcct?ccttggtggc?agctacaacg?ctcgtgaacc??2100

agcacacgac?ggctaaggtt?gtggagaggg?ttgtgcaaaa?tgtgtcatat?attgctcaaa??2160

cccaggacca?attcacccac?ctgttcagga?atataaacaa?cagattaaat?gtcctacacc??2220

atagagtttc?atacttggag?tgtacactta?atgggaatga?aagaccccac?ctgtaggttt??2280

ggcaagctag?gatcaaggtt?aggaacagag?agacagcaga?atatgggcca?aacaggatat??2340

ctgtggtaag?cagttcctgc?cccggctcag?ggccaagaac?agttggaaca?gcagaatatg??2400

ggccaaacag?gatatctgtg?gtaagcagtt?cctgccccgg?ctcagggcca?agaacagatg??2460

gtccccagat?gcggtcccgc?cctcagcagt?ttctagagaa?ccatcagatg?tttccagggt??2520

gccccaagga?cctgaaatga?ccctgtgcct?tatttgaact?aaccaatcag?ttcgcttctc??2580

gcttctgttc?gcgcgcttct?gctccccgag?ctctatataa?gcagagctcg?tttagtgaac??2640

cgtcagatcg?cctggagacg?ccatccacgc?tgttttgacc?tccatagaag?acaccgactc??2700

tagaggatcc?accggtcgcc?acttaaggcc?tgcagctagc?accggttgta?caagtcaagc??2760

ggccaaccct?ccctagatct?gttaatcaac?ctctggatta?caaaatttgt?gaaagattga??2820

ctggtattct?taactatgtt?gctcctttta?cgctatgtgg?atacgctgct?ttaatgcctt??2880

tgtatcatgc?tattgcttcc?cgtatggctt?tcattttctc?ctccttgtat?aaatcctggt??2940

tgctgtctct?ttatgaggag?ttgtggcccg?ttgtcaggca?acgtggcgtg?gtgtgcactg??3000

tgtttgctga?cgcaaccccc?actggttggg?gcattgccac?cacctgtcag?ctcctttccg??3060

ggactttcgc?tttccccctc?cctattgcca?cggcggaact?catcgccgcc?tgccttgccc??3120

gctgctggac?aggggctcgg?ctgttgggca?ctgacaattc?cgtggtgttg?tcggggaagc??3180

tgacgtcctt?tccatggctg?ctcgcctgtg?ttgccacctg?gattctgcgc?gggacgtcct??3240

tctgctacgt?cccttcggcc?ctcaatccag?cggaccttcc?ttcccgcggc?ctgctgccgg??3300

ctctgcggcc?tcttccgcgt?cttcgccttc?gccctcagac?gagtcggatc?tccctttggg??3360

ccgcctcccc?gcctgtttcg?ccttctagga?aactcctttg?ggacatcttc?cgccacgctc??3420

ctattttaaa?cttaaaaggg?tggactgtgg?ggcagggtgg?gacctcagga?caacagcagc??3480

ccccggactt?cccatatgtg?tttatttgtg?aaatttgtga?tgctattgct?ttatttgtaa??3540

tctgtacttc?agctcgtgta?gctcattagc?tccgagctcc?ccaacctaca?gcctgagagg??3600

cactggctcg?gttgggtagc?cagcctttcg?ggtaataaag?gcttgttggc?attcggcatc??3660

tacccgtgcc?tcctgtcttg?tcttactcga?gcgaacccac?aactccgtcc?tgctgagctc??3720

acagctcgcg?gggcggtgaa?gaacacccaa?cagatatata?ctgtcaacat?cccatttggt??3780

agcttatgtt?ctagacaaga?ttctcaacaa?attcttcccc?ctgaatgttt?atttaaaaaa??3840

aaaaaacaac?tactagggct?ctgtgcatat?gtaagtgaga?tccttattag?caggagaaca??3900

gcaataagat?attattacat?tacaatatta?tatcctaggg?tattataatg?caaggccatt??3960

atcacatact?tggctaacag?ggtccatact?gttgtaatgt?attaaaacca?gactgagtaa??4020

taaaattgac?aacaatataa?tcatcatctt?tgttataggt?gggggcattt?ttcagatgag??4080

gtctcagagc?acctgccaag?catggacctc?gagggggggc?ccggtaccca?gcttttgttc??4140

cctttagtga?gggttaattg?cgcgcttggc?gtaatcatgg?tcatagctgt?ttcctgtgtg??4200

aaattgttat?ccgctcacaa?ttccacacaa?catacgagcc?gggagcataa?agtgtaaagc??4260

ctggggtgcc?taatgagtga?gctaactcac?attaattgcg?ttgcgctcac?tgcccgcttt??4320

ccagtcggga?aacctgtcgt?gccagctgca?ttaatgaatc?ggccaacgcg?cggggagagg??4380

cggtttgcgt?attgggcgct?cttccgcttc?ctcgctcact?gactcgctgc?gctcggtcgt??4440

tcggctgcgg?cgagcggtat?cagctcactc?aaaggcggta?atacggttat?ccacagaatc??4500

aggggataac?gcaggaaaga?acatgtgagc?aaaaggccag?caaaaggcca?ggaaccgtaa??4560

aaaggccgcg?ttgctggcgt?ttttccatag?gctccgcccc?cctgacgagc?atcacaaaaa??4620

tcgacgctca?agtcagaggt?ggcgaaaccc?gacaggacta?taaagatacc?aggcgtttcc??4680

ccctggaagc?tccctcgtgc?gctctcctgt?tccgaccctg?ccgcttaccg?gatacctgtc??4740

cgcctttctc?ccttcgggaa?gcgtggcgct?ttctcatagc?tcacgctgta?ggtatctcag??4800

ttcggtgtag?gtcgttcgct?ccaagctggg?ctgtgtgcac?gaaccccccg?ttcagcccga??4860

ccgctgcgcc?ttatccggta?actatcgtct?tgagtccaac?ccggtaagac?acgacttatc??4920

gccactggca?gcagccactg?gtaacaggat?tagcagagcg?aggtatgtag?gcggtgctac??4980

agagttcttg?aagtggtggc?ctaactacgg?ctacactaga?agaacagtat?ttggtatctg??5040

cgctctgctg?aagccagtta?ccttcggaaa?aagagttggt?agctcttgat?ccggcaaaca??5100

aaccaccgct?ggtagcggtg?gtttttttgt?ttgcaagcag?cagattacgc?gcagaaaaaa??5160

aggatctcaa?gaagatcctt?tgatcttttc?tacggggtct?gacgctcagt?ggaacgaaaa??5220

ctcacgttaa?gggattttgg?tcatgagatt?atcaaaaagg?atcttcacct?agatcctttt??5280

aaattaaaaa?tgaagtttta?aatcaatcta?aagtatatat?gagtaaactt?ggtctgacag??5340

ttaccaatgc?ttaatcagtg?aggcacctat?ctcagcgatc?tgtctatttc?gttcatccat??5400

agttgcctga?ctccccgtcg?tgtagataac?tacgatacgg?gagggcttac?catctggccc??5460

cagtgctgca?atgataccgc?gagacccacg?ctcaccggct?ccagatttat?cagcaataaa??5520

ccagccagcc?ggaagggccg?agcgcagaag?tggtcctgca?actttatccg?cctccatcca??5580

gtctattaat?tgttgccggg?aagctagagt?aagtagttcg?ccagttaata?gtttgcgcaa??5640

cgttgttgcc?attgctacag?gcatcgtggt?gtcacgctcg?tcgtttggta?tggcttcatt??5700

cagctccggt?tcccaacgat?caaggcgagt?tacatgatcc?cccatgttgt?gcaaaaaagc??5760

ggttagctcc?ttcggtcctc?cgatcgttgt?cagaagtaag?ttggccgcag?tgttatcact??5820

catggttatg?gcagcactgc?ataattctct?tactgtcatg?ccatccgtaa?gatgcttttc??5880

tgtgactggt?gagtactcaa?ccaagtcatt?ctgagaatag?tgtatgcggc?gaccgagttg??5940

ctcttgcccg?gcgtcaatac?gggataatac?cgcgccacat?agcagaactt?taaaagtgct??6000

catcattgga?aaacgttctt?cggggcgaaa?actctcaagg?atcttaccgc?tgttgagatc??6060

cagttcgatg?taacccactc?gtgcacccaa?ctgatcttca?gcatctttta?ctttcaccag??6120

cgtttctggg?tgagcaaaaa?caggaaggca?aaatgccgca?aaaaagggaa?taagggcgac??6180

acggaaatgt?tgaatactca?tactcttcct?ttttcaatat?tattgaagca?tttatcaggg??6240

ttattgtctc?atgagcggat?acatatttga?atgtatttag?aaaaataaac?aaataggggt??6300

tccgcgcaca?tttccccgaa?aagtgccac????????????????????????????????????6329

<210>23

<211>7068

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic polyribonucleotides

<400>23

ctaaattgta?agcgttaata?ttttgttaaa?attcgcgtta?aatttttgtt?aaatcagctc????60

attttttaac?caataggccg?aaatcggcaa?aatcccttat?aaatcaaaag?aatagaccga???120

gatagggttg?agtgttgttc?cagtttggaa?caagagtcca?ctattaaaga?acgtggactc???180

caacgtcaaa?gggcgaaaaa?ccgtctatca?gggcgatggc?ccactacgtg?aaccatcacc???240

ctaatcaagt?tttttggggt?cgaggtgccg?taaagcacta?aatcggaacc?ctaaagggag???300

cccccgattt?agagcttgac?ggggaaagcc?ggcgaacgtg?gcgagaaagg?aagggaagaa???360

agcgaaagga?gcgggcgcta?gggcgctggc?aagtgtagcg?gtcacgctgc?gcgtaaccac???420

cacacccgcc?gcgcttaatg?cgccgctaca?gggcgcgtcc?cattcgccat?tcaggctgcg???480

caactgttgg?gaagggcgat?cggtgcgggc?ctcttcgcta?ttacgccagc?tggcgaaagg???540

gggatgtgct?gcaaggcgat?taagttgggt?aacgccaggg?ttttcccagt?cacgacgttg???600

taaaacgacg?gccagtgagc?gcgcgtaata?cgactcacta?tagggcgaat?tggagctcca???660

ccgcggtggc?ggccgctcta?gaactagtgg?atcccccggc?atcccgtagt?tattaatagt???720

aatcaattac?ggggtcatta?gttcatagcc?catatatgga?gttccgcgtt?acataactta???780

cggtaaatgg?cccgcctggc?tgaccgccca?acgacccccg?cccattgacg?tcaataatga???840

cgtatgttcc?catagtaacg?ccaataggga?ctttccattg?acgtcaatgg?gtggagtatt???900

tacggtaaac?tgcccacttg?gcagtacatc?aagtgtatca?tatgccaagt?ccgcccccta???960

ttgacgtcaa?tgacggtaaa?tggcccgcct?ggcattatgc?ccagtacatg?accttacggg??1020

actttcctac?ttggcagtac?atctacgtat?tagtcatcgc?tattaccatg?gtgatgcggt??1080

tttggcagta?caccaatggg?cgtggatagc?ggtttgactc?acggggattt?ccaagtctcc??1140

accccattga?cgtcaatggg?agtttgtttt?ggcaccaaaa?tcaacgggac?tttccaaaat??1200

gtcgtaataa?ccccgccccg?ttgacgcaaa?tgggcggtag?gcgtgtacgg?tgggaggtct??1260

atataaacca?tatcttcact?ctgtacttca?gctcgtgtag?ctcattagct?ccgagctccc??1320

caacctacag?cctgagaggc?actggctcgg?ttgggtagcc?agcctttcgg?gtaataaagg??1380

cttgttggca?ttcggcatct?acccgtgcct?cctgtcttgt?cttactcgag?cgaacccaca??1440

actccgtcct?gctgagctca?cagctcgcgg?ggcggtgaag?aacacccaac?agttggcgcc??1500

caacgtgggg?ctgagtaaga?gagactcggc?tcgagtaaaa?gaagacccag?ctcgaacgag??1560

aagactccgg?acaggtgagt?agttgcgtgt?tttccccggg?gtgaagagaa?gggagttaga??1620

aaagaagctt?cgtaaggtta?gggtgacacc?ccaacaggat?aaatattata?ctatagggaa??1680

tcttcaatgg?gccattagaa?tcacgtgatg?catcgataaa?taaaaaaaga?gggggaatag??1740

ggggccatac?accatatgaa?atatacctag?aatcagaaca?taccaaatac?caagaccaac??1800

tagaacaaca?attttcaaaa?caaaaaattg?aaaagtggtg?ttacgtaagg?aacagaagaa??1860

aggaatggaa?aggaccctac?aaagtgttgt?gggacggaga?cggggcagca?gtaatagagg??1920

agaattcgtg?gattcttgta?aaggtcccca?gctatgggtt?tgtggtagta?aatgacacag??1980

atacaccacc?atccctccgc?atccgaaagc?ctcgagcagt?cggactagca?atattcctgc??2040

ttgtgctggc?tatcatggcc?atcacatcct?ccttggtggc?agctacaacg?ctcgtgaacc??2100

agcacacgac?ggctaaggtt?gtggagaggg?ttgtgcaaaa?tgtgtcatat?attgctcaaa??2160

cccaggacca?attcacccac?ctgttcagga?atataaacaa?cagattaaat?gtcctacacc??2220

atagagtttc?atacttggag?tgtacactta?atgggaatga?aagaccccac?ctgtaggttt??2280

ggcaagctag?gatcaaggtt?aggaacagag?agacagcaga?atatgggcca?aacaggatat??2340

ctgtggtaag?cagttcctgc?cccggctcag?ggccaagaac?agttggaaca?gcagaatatg??2400

ggccaaacag?gatatctgtg?gtaagcagtt?cctgccccgg?ctcagggcca?agaacagatg??2460

gtccccagat?gcggtcccgc?cctcagcagt?ttctagagaa?ccatcagatg?tttccagggt??2520

gccccaagga?cctgaaatga?ccctgtgcct?tatttgaact?aaccaatcag?ttcgcttctc??2580

gcttctgttc?gcgcgcttct?gctccccgag?ctctatataa?gcagagctcg?tttagtgaac??2640

cgtcagatcg?cctggagacg?ccatccacgc?tgttttgacc?tccatagaag?acaccgactc??2700

tagaggatcc?accggtcgcc?acttaaggcc?cggtcgccac?catggtgagc?aagggcgagg??2760

agctgttcac?cggggtggtg?cccatcctgg?tcgagctgga?cggcgacgta?aacggccaca??2820

agttcagcgt?gtccggcgag?ggcgagggcg?atgccaccta?cggcaagctg?accctgaagt??2880

tcatctgcac?caccggcaag?ctgcccgtgc?cctggcccac?cctcgtgacc?accctgacct??2940

acggcgtgca?gtgcttcagc?cgctaccccg?accacatgaa?gcagcacgac?ttcttcaagt??3000

ccgccatgcc?cgaaggctac?gtccaggagc?gcaccatctt?cttcaaggac?gacggcaact??3060

acaagacccg?cgccgaggtg?aagttcgagg?gcgacaccct?ggtgaaccgc?atcgagctga??3120

agggcatcga?cttcaaggag?gacggcaaca?tcctggggca?caagctggag?tacaactaca??3180

acagccacaa?cgtctatatc?atggccgaca?agcagaagaa?cggcatcaag?gtgaacttca??3240

agatccgcca?caacatcgag?gacggcagcg?tgcagctcgc?cgaccactac?cagcagaaca??3300

cccccatcgg?cgacggcccc?gtgctgctgc?ccgacaacca?ctacctgagc?acccagtccg??3360

ccctgagcaa?agaccccaac?gagaagcgcg?atcacatggt?cctgctggag?ttcgtgaccg??3420

ccgccgggat?cactctcggc?atggacgagc?tgtacaagta?aagtcgacct?gcagctagca??3480

ccggttgtac?aagtcaagcg?gccaaccctc?cctagatctg?ttaatcaacc?tctggattac??3540

aaaatttgtg?aaagattgac?tggtattctt?aactatgttg?ctccttttac?gctatgtgga??3600

tacgctgctt?taatgccttt?gtatcatgct?attgcttccc?gtatggcttt?cattttctcc??3660

tccttgtata?aatcctggtt?gctgtctctt?tatgaggagt?tgtggcccgt?tgtcaggcaa??3720

cgtggcgtgg?tgtgcactgt?gtttgctgac?gcaaccccca?ctggttgggg?cattgccacc??3780

acctgtcagc?tcctttccgg?gactttcgct?ttccccctcc?ctattgccac?ggcggaactc??3840

atcgccgcct?gccttgcccg?ctgctggaca?ggggctcggc?tgttgggcac?tgacaattcc??3900

gtggtgttgt?cggggaagct?gacgtccttt?ccatggctgc?tcgcctgtgt?tgccacctgg??3960

attctgcgcg?ggacgtcctt?ctgctacgtc?ccttcggccc?tcaatccagc?ggaccttcct??4020

tcccgcggcc?tgctgccggc?tctgcggcct?cttccgcgtc?ttcgccttcg?ccctcagacg??4080

agtcggatct?ccctttgggc?cgcctccccg?cctgtttcgc?cttctaggaa?actcctttgg??4140

gacatcttcc?gccacgctcc?tattttaaac?ttaaaagggt?ggactgtggg?gcagggtggg??4200

acctcaggac?aacagcagcc?cccggacttc?ccatatgtgt?ttatttgtga?aatttgtgat??4260

gctattgctt?tatttgtaat?ctgtacttca?gctcgtgtag?ctcattagct?ccgagctccc??4320

caacctacag?cctgagaggc?actggctcgg?ttgggtagcc?agcctttcgg?gtaataaagg??4380

cttgttggca?ttcggcatct?acccgtgcct?cctgtcttgt?cttactcgag?cgaacccaca??4440

actccgtcct?gctgagctca?cagctcgcgg?ggcggtgaag?aacacccaac?agatatatac??4500

tgtcaacatc?ccatttggta?gcttatgttc?tagacaagat?tctcaacaaa?ttcttccccc??4560

tgaatgttta?tttaaaaaaa?aaaaacaact?actagggctc?tgtgcatatg?taagtgagat??4620

ccttattagc?aggagaacag?caataagata?ttattacatt?acaatattat?atcctagggt??4680

attataatgc?aaggccatta?tcacatactt?ggctaacagg?gtccatactg?ttgtaatgta??4740

ttaaaaccag?actgagtaat?aaaattgaca?acaatataat?catcatcttt?gttataggtg??4800

ggggcatttt?tcagatgagg?tctcagagca?cctgccaagc?atggacctcg?agggggggcc??4860

cggtacccag?cttttgttcc?ctttagtgag?ggttaattgc?gcgcttggcg?taatcatggt??4920

catagctgtt?tcctgtgtga?aattgttatc?cgctcacaat?tccacacaac?atacgagccg??4980

ggagcataaa?gtgtaaagcc?tggggtgcct?aatgagtgag?ctaactcaca?ttaattgcgt??5040

tgcgctcact?gcccgctttc?cagtcgggaa?acctgtcgtg?ccagctgcat?taatgaatcg??5100

gccaacgcgc?ggggagaggc?ggtttgcgta?ttgggcgctc?ttccgcttcc?tcgctcactg??5160

actcgctgcg?ctcggtcgtt?cggctgcggc?gagcggtatc?agctcactca?aaggcggtaa??5220

tacggttatc?cacagaatca?ggggataacg?caggaaagaa?catgtgagca?aaaggccagc??5280

aaaaggccag?gaaccgtaaa?aaggccgcgt?tgctggcgtt?tttccatagg?ctccgccccc??5340

ctgacgagca?tcacaaaaat?cgacgctcaa?gtcagaggtg?gcgaaacccg?acaggactat??5400

aaagatacca?ggcgtttccc?cctggaagct?ccctcgtgcg?ctctcctgtt?ccgaccctgc??5460

cgcttaccgg?atacctgtcc?gcctttctcc?cttcgggaag?cgtggcgctt?tctcatagct??5520

cacgctgtag?gtatctcagt?tcggtgtagg?tcgttcgctc?caagctgggc?tgtgtgcacg??5580

aaccccccgt?tcagcccgac?cgctgcgcct?tatccggtaa?ctatcgtctt?gagtccaacc??5640

cggtaagaca?cgacttatcg?ccactggcag?cagccactgg?taacaggatt?agcagagcga??5700

ggtatgtagg?cggtgctaca?gagttcttga?agtggtggcc?taactacggc?tacactagaa??5760

gaacagtatt?tggtatctgc?gctctgctga?agccagttac?cttcggaaaa?agagttggta??5820

gctcttgatc?cggcaaacaa?accaccgctg?gtagcggtgg?tttttttgtt?tgcaagcagc??5880

agattacgcg?cagaaaaaaa?ggatctcaag?aagatccttt?gatcttttct?acggggtctg??5940

acgctcagtg?gaacgaaaac?tcacgttaag?ggattttggt?catgagatta?tcaaaaagga??6000

tcttcaccta?gatcctttta?aattaaaaat?gaagttttaa?atcaatctaa?agtatatatg??6060

agtaaacttg?gtctgacagt?taccaatgct?taatcagtga?ggcacctatc?tcagcgatct??6120

gtctatttcg?ttcatccata?gttgcctgac?tccccgtcgt?gtagataact?acgatacggg??6180

agggcttacc?atctggcccc?agtgctgcaa?tgataccgcg?agacccacgc?tcaccggctc??6240

cagatttatc?agcaataaac?cagccagccg?gaagggccga?gcgcagaagt?ggtcctgcaa??6300

ctttatccgc?ctccatccag?tctattaatt?gttgccggga?agctagagta?agtagttcgc??6360

cagttaatag?tttgcgcaac?gttgttgcca?ttgctacagg?catcgtggtg?tcacgctcgt??6420

cgtttggtat?ggcttcattc?agctccggtt?cccaacgatc?aaggcgagtt?acatgatccc??6480

ccatgttgtg?caaaaaagcg?gttagctcct?tcggtcctcc?gatcgttgtc?agaagtaagt??6540

tggccgcagt?gttatcactc?atggttatgg?cagcactgca?taattctctt?actgtcatgc??6600

catccgtaag?atgcttttct?gtgactggtg?agtactcaac?caagtcattc?tgagaatagt??6660

gtatgcggcg?accgagttgc?tcttgcccgg?cgtcaatacg?ggataatacc?gcgccacata??6720

gcagaacttt?aaaagtgctc?atcattggaa?aacgttcttc?ggggcgaaaa?ctctcaagga??6780

tcttaccgct?gttgagatcc?agttcgatgt?aacccactcg?tgcacccaac?tgatcttcag??6840

catcttttac?tttcaccagc?gtttctgggt?gagcaaaaac?aggaaggcaa?aatgccgcaa??6900

aaaagggaat?aagggcgaca?cggaaatgtt?gaatactcat?actcttcctt?tttcaatatt??6960

attgaagcat?ttatcagggt?tattgtctca?tgagcggata?catatttgaa?tgtatttaga??7020

aaaataaaca?aataggggtt?ccgcgcacat?ttccccgaaa?agtgccac???????????????7068

<210>24

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic primer

<400>24

acaccaccat?ccctccgcat?ccga???????????????????????????????????????????24

<210>25

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic primer

<400>25

tgggtttgtg?gtagtaaatg?acac???????????????????????????????????????????24

<210>26

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉artificial sequence note: synthetic primer

<400>26

tggttcacga?gcgttgtagc????????????????????????????????????????????????20

<210>27

<211>253

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>27

Met?His?Ser?Trp?Glu?Arg?Leu?Ala?Val?Leu?Val?Leu?Leu?Gly?Ala?Ala

1???????????????5???????????????????10??????????????????15

Ala?Cys?Ala?Ala?Pro?Pro?Arg?Gly?Arg?Ile?Leu?Gly?Gly?Arg?Glu?Ala

20??????????????????25??????????????????30

Glu?Ala?His?Ala?Arg?Pro?Tyr?Met?Ala?Ser?Val?Gln?Leu?Asn?Gly?Ala

35??????????????????40??????????????????45

His?Leu?Cys?Gly?Gly?Val?Leu?Val?Ala?Glu?Gln?Trp?Val?Leu?Ser?Ala

50?????????????????55?????????????????60

Ala?His?Cys?Leu?Glu?Asp?Ala?Ala?Asp?Gly?Lys?Val?Gln?Val?Leu?Leu

65?????????????????70?????????????????75?????????????????80

Gly?Ala?His?Ser?Leu?Ser?Gln?Pro?Glu?Pro?Ser?Lys?Arg?Leu?Tyr?Asp

85??????????????????90??????????????????95

Val?Leu?Arg?Ala?Val?Pro?His?Pro?Asp?Ser?Gln?Pro?Asp?Thr?Ile?Asp

100?????????????????105?????????????????110

His?Asp?Leu?Leu?Leu?Leu?Gln?Leu?Ser?Glu?Lys?Ala?Thr?Leu?Gly?Pro

115?????????????????120?????????????????125

Ala?Val?Arg?Pro?Leu?Pro?Trp?Gln?Arg?Val?Asp?Arg?Asp?Val?Ala?Pro

130?????????????????135?????????????????140

Gly?Thr?Leu?Cys?Asp?Val?Ala?Gly?Trp?Gly?Ile?Val?Asn?His?Ala?Gly

145?????????????????150?????????????????155?????????????????160

Arg?Arg?Pro?Asp?Ser?Leu?Gln?His?Val?Leu?Leu?Pro?Val?Leu?Asp?Arg

165?????????????????170?????????????????175

Ala?Thr?Cys?Asn?Arg?Arg?Thr?His?His?Asp?Gly?Ala?Ile?Thr?Glu?Arg

180?????????????????185?????????????????190

Leu?Met?Cys?Ala?Glu?Ser?Asn?Arg?Arg?Asp?Ser?Cys?Lys?Gly?Asp?Ser

195?????????????????200?????????????????205

Gly?Gly?Pro?Leu?Val?Cys?Gly?Gly?Val?Leu?Glu?Gly?Val?Val?Thr?Ser

210?????????????????215?????????????????220

Gly?Ser?Arg?Val?Cys?Gly?Asn?Arg?Lys?Lys?Pro?Gly?Ile?Tyr?Thr?Arg

225?????????????????230?????????????????235?????????????????240

Val?Ala?Ser?Tyr?Ala?Ala?Trp?Ile?Asp?Ser?Val?Leu?Ala

245?????????????????250

Claims (47)

1. method for the treatment of the disease of complement-mediated, described method comprise that described pharmaceutical composition comprises to patient's drug administration composition of needs treatment:

(i) the complement factor B analog of inhibition or reduction complement activity;

(ii) the encode carrier of described complement factor B analog;

(iii) suppress or reduce the Complement Factor D analog of complement activity; Or

(iv) the encode carrier of described Complement Factor D analog.

2. the method for claim 1, wherein described carrier is retroviral vector, slow virus carrier, adenovirus vector, herpesvirus vector, hepatitis viruse carrier, SV40 carrier or EBV carrier.

3. method as claimed in claim 2, wherein, described slow virus is HIV, EIAV, SIV or FIV.

4. method as claimed in claim 2, wherein, described slow virus is BIV.

5. the method for claim 1, wherein described carrier is adeno-associated virus (AAV) carrier.

6. the method for claim 1, wherein described complement factor B analog comprises the C3b binding affinity of comparing increase with natural complement factor B; With

(i) compare the proteinase activity that weakens with natural complement factor B; Or

(ii) compare the ability of being cut that weakens by factor D with natural complement factor B.

7. the method for claim 1, wherein described complement factor B analog has to be compared the proteinase activity that weakens and compares the affinity to C3b that does not significantly increase with natural complement factor B with natural complement factor B.

8. the method for claim 1, wherein described complement factor B analog comprises change at C3b in conjunction with the territory.

9. method as claimed in claim 8, wherein, C3b comprises in conjunction with the described change in the territory:

(i) replacement of aspartic acid and/or asparagine or disappearance; Or

(ii) be close to the insertion of described aspartic acid or described asparagine,

Wherein said aspartic acid corresponding to 279 amino acids of SEQ ID NO:2 and

Wherein said asparagine is corresponding to 285 amino acids of SEQ ID NO:2.

10. method as claimed in claim 9, wherein, described aspartic acid and/or described asparagine are by another aminoacid replacement.

11. method as claimed in claim 9, wherein, described aspartic acid is replaced by glycine, alanine or asparagine.

12. method as claimed in claim 9, wherein, described asparagine is replaced by glycine, alanine or aspartic acid.

13. method as claimed in claim 9, wherein, described replacement comprises with glycine and substitutes described aspartic acid and substitute described asparagine with aspartic acid.

14. method as claimed in claim 6, wherein, described complement factor B analog is included in the change in the factor D cleavage site.

15. method as claimed in claim 14, wherein, the described change in the factor D cleavage site comprises:

(i) at least one lysine or at least one arginic replacement or disappearance; Or

(ii) be close to described at least one lysine or described at least one arginic insertion,

Wherein said at least one lysine corresponding to 258 of SEQ ID NO:2 or 260 amino acids and

Wherein said at least one asparagine is corresponding to 259 amino acids of SEQ ID NO:2.

16. method as claimed in claim 15, wherein, described change comprises at least one lysine and/or at least one arginic replacement.

17. method as claimed in claim 15, wherein, described replacement comprises with alanine and substitutes described at least one lysine and substitute described at least one arginine with alanine.

18. method as claimed in claim 15 wherein, is replaced by alanine separately corresponding to the described amino acid of 258～260 amino acids of SEQ ID NO:2.

19. the method for claim 1, wherein described complement factor B analog comprises:

(i) 26～764 amino acids of SEQ ID NO:4 or SEQ ID NO:4;

(ii) 26～764 amino acids of SEQ ID NO:6 or SEQ ID NO:6; Or

(iii) 26～764 amino acids of SEQ ID NO:8 or SEQ ID NO:8.

20. the method for claim 1, wherein described Complement Factor D analog comprises the ability of comparing the cutting factor B of reduction with natural Complement Factor D.

21. method as claimed in claim 20, wherein, described Complement Factor D analog is included in the change in the serine protease catalytic domain of natural Complement Factor D.

22. method as claimed in claim 21, wherein, the change in the serine protease catalytic domain of described natural Complement Factor D comprises:

(i) corresponding to amino acid whose replacement or the disappearance of His66, Asp114 or the Ser208 of SEQ ID NO:27; Or

(ii) insert at least one amino acid corresponding to His66, the Asp114 of the SEQ ID NO:27 of natural Complement Factor D or the amino acid place of Ser208 the next-door neighbour.

23. method as claimed in claim 22, wherein

(i) be used as neutral amino acid, electronegative amino acid or at least one aminoacid replacement of nonpolar amino acid corresponding to the amino acid of His66;

(ii) be used as neutral amino acid, positively charged amino acid or at least one aminoacid replacement of nonpolar amino acid corresponding to the amino acid of Asp114; Or

(iii) be used as at least one aminoacid replacement of charged amino acid or nonpolar amino acid corresponding to the amino acid of Ser208.

24. method as claimed in claim 20, wherein, described Complement Factor D analog is compared with natural Complement Factor D at the N end and is comprised one or more additional amino acids.

25. method as claimed in claim 24, wherein, described one or more additional amino acids comprise glycine and arginine.

26. the method for claim 1, wherein the disease of described complement-mediated is an eye disease.

27. method as claimed in claim 26, wherein, described pharmaceutical composition is transported to eye.

28. method as claimed in claim 27, wherein, described pharmaceutical composition is by injection, cornea local injection in intravitreal injection, subretinal injection, the camera oculi anterior or use, inject under the subconjunctival injection, capsula bulbi or put drops in one's eyes and carry.

29. the method for claim 1, wherein, described disease be macular degeneration, senile macular degeneration (AMD), pattern atrophy, moist AMD, myocardial infarction, dryness AMD, drusen formation, palsy, ischemic damage and reperfusion damage, BDR, vitreoretinopathy, traumatic organ damage, cornea inflammation, uveitis, high intraocular pressure or glaucoma.

30. the method for claim 1, described method also are included in and use before the described pharmaceutical composition, simultaneously or afterwards the patient is used complement inhibitor or anti-angiogenesis.

31. method as claimed in claim 30, wherein, described complement inhibitor is selected from the group of being made up of the soluble form of factor H, factor H sample 1, MCP, DAF or MCP.

32. the method for claim 1, wherein before using described pharmaceutical composition, simultaneously or use antiinflammatory agent afterwards.

33. method as claimed in claim 32, wherein, described antiinflammatory agent

(i) with described pharmaceutical composition use simultaneously or

(ii) described pharmaceutical composition comprises described antiinflammatory agent.

34. method as claimed in claim 32, wherein, described antiinflammatory agent is applied to eye.

35. method as claimed in claim 32, wherein, described antiinflammatory agent is selected from the group of being made up of sodium sulfo benzoate, dexamethasone sodium phosphate, fluorometholone, bromfenac, pranoprofen, RESTASISTM, cyclosporin ophthalmic emulsion, naproxen, glucocorticoid, ketorolac, brufen, Tolmetin, NSAID (non-steroidal anti-inflammatory drug), steroidal anti-inflammatory medicine, Diclofenac, Flurbiprofen, Indomethacin and suprofen between dexamethasone, dexamethasone.

36. being viral vectors and described viral vectors, the method for claim 1, wherein said carrier comprise decay accelerating factor.

37. the viral vectors of the complement factor B analog of encoding, wherein, described complement factor B analog suppresses or the reduction complement activity.

38. viral vectors as claimed in claim 35, wherein, described complement factor B analog comprises the C3b binding affinity of comparing increase with natural complement factor B; With

(i) compare the proteinase activity that weakens with natural complement factor B; Or

(ii) compare the ability of being cut that weakens by factor D with natural complement factor B.

39. cell that produces the described viral vectors of claim 38.

40. the viral vectors of the Complement Factor D analog of encoding, wherein, described Complement Factor D analog suppresses or the reduction complement activity.

41. cell that produces the described viral vectors of claim 40.

42. a pharmaceutical composition, described pharmaceutical composition comprise the complement factor B analog of the C3b binding affinity with the proteinase activity that weakens and change.

43. a Complement Factor D analog, described Complement Factor D analog are compared the ability of the cutting factor B with reduction with natural Complement Factor D.

44. a composition that comprises complement factor B analog and/or Complement Factor D analog, wherein, the purity of described factor B analog or described factor D analog is at least 95%～99%.

45. a pharmaceutical composition, described pharmaceutical composition comprise the described Complement Factor D analog of claim 43.

46. as claim 42 or 45 described pharmaceutical compositions, described pharmaceutical composition comprises at least a composition that is selected from the group of being made up of histidine, MgCl2, trehalose, polysorbate, polysorbate 20 and NaCl.

47. a pharmaceutical composition, described pharmaceutical composition comprises:

(i) the complement factor B analog of inhibition or reduction complement activity;

(ii) the encode carrier of described complement factor B analog;

(iii) suppress or reduce the Complement Factor D analog of complement activity; Or

(iv) the encode carrier of described Complement Factor D analog.

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