Mixed polypeptide vaccine and preparation thereof and applicationTechnical field
The present invention relates to medicinal preparation, relate in particular to a kind of mixed polypeptide vaccine and preparation thereof that can suppress autoimmune disease and produce and use.
Technical background
Protein generally is made up of with upper amino acid 50-80, the chemical compound with certain three dimensions conformation.Polypeptide: generally form chemical compound with certain three dimensions conformation by 50 following aminoacid.Little peptide: form by 30 following aminoacid.Generally do not have the three dimensions conformation.Cell chemotactic factor: secretedly in the animal body can induce various leukokinetic protein.Antigen: cause that animal produces immunoreation, comprises the material that produces antibody.The general all materials of right and wrong self of antigen.Autoantigen: cause that animal produces immunoreation, comprises the material of the health self that produces antibody.The autoimmune toleration: under the normal condition, self material do not produced immunoreation in the body and do not produce the antibody of anti-self material, this mechanism is called " autoimmune toleration ".Antigenic specificity: be meant mutual anastomose property between the material or specific aim, specificity.CpG: the distinctive nucleotide sequence of antibacterial, nucleotide is not wherein methylated.The toxin of antibacterial: some antibacterials secrete some special protein, these protein can cause the untoward reaction that human body is huge, these protein molecular weights are all very big, often be made up of several different subunits, its whole molecule of the excretory tetanus toxin of analogy clostridium tetani is made up of 1315 aminoacid.Covalently bound: the connection between chemical compound is by the covalent bond of chemistry definition, comprises peptide bond, disulfide bridge bond etc.
World name and abbreviation about chemotactic factor:
MCP-1：monocyte?chemoattractant?protein?1；
MCP-3：monocyte?chemoattractant?protein?3；
MCP-4：monocyte?chemoattractant?protein?4；
MCP-5：monocyte?chemoattractant?protein?5；
MIP-1?alpha：macrophage?inflammatory?protein?1?alpha；
RANTES：regulated?on?activation，normal?T-cell?expressed?and
secreted；
IL-8:Interleukin 8, people's chemotactic factor;
IP-10：IFN-gamma?inducible?protein?10；
MIG：monokine?induced?by?IFN-gamma；
ITAC：interferon-inducible?T?cell?alpha?chemoattractant；
GRO-alpha：growth?related?oncogene?alpha；
SLC is 6Ckine:6-cysteine chemokine;
ELC is MIP-3 beta:macrophage inflammatory protein 3 beta;
ENA-78：epithelial?cell-derived?neutrophil?activating?protein?78；
MIP-2:monocyte inflammatory protein-2, the chemotactic factor of Mus;
Remove particularly point out kind beyond, above-mentioned chemotactic factor all is present in people and the Mus body.The chemotactic factor sequence of people and Mus has homology, but inequality.
Autoimmune disease such as rheumatic arthritis, lupus erythematosus, noninfectious colitis, psoriasis, multiple hard flesh diseases etc. all are because the excessive immunoreation of human body causes.Patient since genetic flaw or the day after tomorrow virus/bacterial infection cause immunomodulating out of control, the tissue of human body self and organ are produced immunoreation, cause skeleton, colon, kidney, heart or neural permanent damage.The characteristics of these diseases all are to show effect repeatedly, constantly worsen, and the course of disease is very long, the case decades-long that has, even cannot be cured all one's life, finally cause the syndrome of intractable owing to the heavy damage of body tissue, even threat to life or formation canceration.For example scleroderma claims systemic sclerosis again, is to be the connective tissue disease of characteristics with limitation or diffusivity skin and internal organ connective fibrosis, sclerosis and atrophy, and disease progression is dangerous, even threat to life.Part patient's state of an illness is through just occurring the clinical manifestation that vitals such as special internal organs kidney, heart are got involved for a long time.Such as sjogren syndrome, be a kind of to invade the autoimmune disease of lachrymal gland, salivary gland again, it not only invades the external secretion body of gland, cause xerostomia, eye to do, also can invade a plurality of organs of whole body, produce diversified clinical manifestation, be the common rheumatism that is only second to rheumatoid arthritis.Another example is a systemic lupus erythematosus (sle).Nephropathy and heart disease are one of modal clinical manifestations of systemic lupus erythematosus (sle), can cause renal failure and death.Therefore have only and early treatment, just can prevent, could prolong the life cycle of systemic lupus erythematosus (sle).Allosome tissue's graft-rejection also is because the excessive immunoreation of body causes.
Autoimmune disease and organ transplantation the patient generally anti-allosome of long-term dependence repel medicine.No matter Western medicine, Chinese medicine all can not effect a radical cure in the market.For the lighter patient of condition of illness, the activeness that some medicine can control disease at present, mitigation symptoms, but can not prevent recurrence.For being in a bad way, the patient who is difficult to disease controlling with general medicine then must use immunosuppressant.And the immunoreactive medicine of inhibition in the market is all undesirable, and perhaps side effect is very high, and perhaps curative effect is not obvious.And the medicine half-life in vivo is all of short duration, and patient needs repeatedly take medicine every day, can keep curative effect.Such therapeutic modality all makes the patient can't bear the heavy load from still in life economically.Therefore market in urgent need is long-acting, does not have remarkable side effect, and cheap preparation, so that satisfy this class patient to suppressing morbidity, the specific demand of drug use and economic aspect.
Discover that the protein of cell chemotactic factor (Chemokine) plays an important role to the pathological process of many autoimmune diseases.Such as, ulcerative colitis and psoriasis, the main member who causes its pathological lesion is mononuclear cell and neutrophilic granulocyte, and these cells are brought out by some cell chemotactic factors just and invade profit and enter tissue.
Cell chemotactic factor is that animal or human's body is endocrine, can induce various leukokinetic protein.So far find that human body has 40 various kinds of cell chemotactic factors, molecular weight is 7,000-11, about 000.Be divided into C altogether according to its chemical constitution, CC, four families of CXC and CX3C.Chemotactic factor by combine with the specific receptor of leukocyte surface and activated receptor bring into play its inducing function.Except inducing cell moves, chemotactic factor is the calcium ionic current of inducing cell also.In vivo, some chemotactic factors participate in all kinds of inflammatory reactions, and other chemotactic factors participation cells normally move.Chemotactic factor has species specificity, and the excretory chemotactic factor sequence of promptly various animals has homology, but inequality.
Cell chemotactic factor (Chemokine) can roughly be divided into againDerivable cell chemotactic factorWithSubstantiallyCell chemotactic factorTwo big classes.Derivable chemotactic factorCan be by various inflammatory factor abduction deliverings, or strengthen and express.Expression levels is the highest is inducing 300 times that can reach normal condition within back 3 hours.At inflammation disease such as lupus erythematosus, the multiple types scleroderma, psoriasis, rheumatism, and allosome tissue's graft-rejection, the expression of derivable chemotactic factor obviously increases.Thereby derivable cell chemotactic factor is otherwise known asThe inflammatory cell chemotactic factor,Comprise IP-10, Mig, ITAC, IL-8, MCPs (MCP-1,2,3,4,5), RANTES, MIP-1alpha/beta, NEA-78, GRO-alpha/beta, SLC, ELC, BCA-1 or the like.From observed another phenomenon of various diseases be, general expression obviously increases simultaneously is not simple a kind of, but multiple inflammation chemotactic factor.This has illustrated the complexity of disease and the fact of multiple chemotactic factor participation effect simultaneously.Current research shows, the antibody of injecting anti-chemotactic factor for sick animal reaches the effect that neutralizes or offset above-mentioned inflammation chemotactic factor, can the part mitigate the disease.But, use external antibody to do treatment and can induce the patient to produce allergic immune response this antibody, can not prolonged and repeatedly use, so needing to be not suitable for the autoimmune disease of long-term treatment.And animal can not produce immunoreation to self material owing to innately have self tolerance, can not produce anti-self material, as the antibody of self chemotactic factor.
Summary of the invention
In order to overcome the above-mentioned shortcoming that prior art exists, the invention provides a kind of mixed polypeptide vaccine, it can bring out the antibody that produces in the body at self multiple different inflammation chemotactic factor.It is mixed by hybridization peptide not of the same race and forms, and wherein every kind of hybridization peptide all is to be discerned with immunity by the amino terminal fragment of specific inflammation chemotactic factor that fragment is covalently bound to form; The present invention also provides the preparation method of these products, particularly utilizes the preparation method of chemosynthesis, and the method for testing of product; Described mixed polypeptide vaccine is applied to prepare treatment or prevents repulsion that all kinds of autoimmune diseases and prevention of organ transplant cause and the vaccine of graft versus host disease.
The objective of the invention is to realize by following technical scheme.
Mixed polypeptide vaccine of the present invention, it is mixed by multiple hybridization peptide not of the same race and forms, and every kind of hybridization peptide is discerned with immunity by the amino terminal fragment of chemotactic factor that fragment is covalently bound to form; Wherein, the amino terminal fragment of chemotactic factor is the aminoterminal little fragments of peptides of inflammation chemokine protein matter; Immunity identification fragment is polypeptide or the dna fragmentation that immune system can be recognized as the dissident.
Aforesaid mixed polypeptide vaccine, described aminoterminal little fragments of peptides comprise and are positioned at SEQ1 to SEQ17 and corresponding nucleotide SEQ24 to SEQ39 that the little peptide of this section is not for there being active autoantigen.
Aforesaid mixed polypeptide vaccine, described immunity identification fragment comprise and be positioned at SEQ18 to SEQ23 and corresponding nucleic acids sequence SEQ40 to SEQ42, is through selecting or chemical modification and remove the bacteriotoxin fragment of innate toxicity.
Aforesaid mixed polypeptide vaccine, it is characterized in that: the amino terminal fragment of the chemotactic factor of described hybridization peptide is meant that aminoterminal one of inflammation in mammals chemokine protein matter does not have the active fragment of chemotactic factor, this fragment is as autoantigen, and this fragment comprises: the amino terminal fragment that is positioned at the MCP-1 of SEQ1 and 24; Be positioned at the amino terminal fragment of the IP-10 of SEQ2 and 25; Be positioned at the amino terminal fragment of the ITAC of SEQ3 and 26; Be positioned at the amino terminal fragment of the MIG of SEQ4 and 27; Be positioned at the amino terminal fragment of the MCP-3 of SEQ5 and 28; Be positioned at the amino terminal fragment of the MCP-4 of SEQ6 and 29; Be positioned at the amino terminal fragment of the RANTES of SEQ7 and 30; Be positioned at the amino terminal fragment of the MIP-1alpha of SEQ8 and 31; Be positioned at the IL-8 amino terminal fragment of SEQ9 and 32; Be positioned at the amino terminal fragment of the ENA-78 of SEQ10 and 33; Be positioned at the amino terminal fragment of the GRO-alpha of SEQ11 and 34; Be positioned at the amino terminal fragment of the SLC of SEQ12 and 35; Be positioned at the amino terminal fragment of the ELC of SEQ13 and 36, and the muroid chemotactic factor fragment that is positioned at SEQ13-17 and 37-39.
Aforesaid mixed polypeptide vaccine, it is characterized in that: the amino terminal fragment of the chemotactic factor of described hybridization peptide also comprises the fragment or the analog of (in the claim 2 and 4) all sequences in aminoterminal little fragments of peptides and the amino terminal fragment, the homology of this analog reaches 50% of this sequence at least, 60%, 70%.
Aforesaid mixed polypeptide vaccine is characterized in that: described immunity identification fragment is to lose the toxic fragment of original toxin through cutting choosing or chemical modification, and this fragment comprises: the special fragment that is positioned at the tetanus toxin of SEQ18; Or containing the dna fragmentation of CpG, this fragment comprises sequence 19-21; Or be positioned at the special fragment of the cholera bacilli toxin (CT) of SEQ22, SEQ41; Or the special fragment of the thermo-responsive toxin of the Escherichia coli that is positioned at SEQ23, SEQ42 (LT).
Aforesaid mixed polypeptide vaccine is characterized in that: described immunity identification fragment comprises the fragment or the analog of immunity identification fragment (in the claim 3 and 7) all sequences in addition, and the homology of this analog reaches 50%, 60%, 70% of this sequence at least.
The present invention produces the method for preparing the described mixed polypeptide vaccine of claim 1, it is characterized in that, the first step is produced the described various hybridization peptides of preparation claim 1, and its described hybridization peptide is to utilize following method to produce preparation: a, utilize chemical synthesis process production preparation; B or utilize gene recombination technology to produce preparation, it comprises the segmental carrier of amino terminal of construction expression various hybridization peptides as claimed in claim 1 or various chemotactic factors, and the bacterial strain that carries these carriers; C or the method for utilizing chemical method to be connected the amino terminal fragment of claim 1 and 4 described chemotactic factors and claim 1 and 5 CpG-DNA fragment; D or the isolation and purification method preparation by the hybridization peptide; Second step, according to kinds of Diseases and involved specific chemokines of PD stage, select two or more suitable hybridization peptides to mix at these factors, again this mixture is prepared into the method for mixed vaccine.
The application of aforesaid mixed polypeptide vaccine is characterized in that, the dosage form of the vaccine that described mixed polypeptide is made can with following composition one of: the absorption type of injection type, peroral dosage form and skin, nasal cavity, genitals, intestinal.
A kind of mixed polypeptide vaccine of the present invention, it is mixed by multiple hybridization peptide not of the same race and forms, can bring out the antigenic antibody of anti-two or more different self inflammatory factors of mammal secretion in vivo, fragment is covalently bound forms by self inflammatory factor fragment and immunity identification for every kind of hybridization peptide; Wherein, self inflammatory factor fragment is the proteinic little fragments of peptides of various inflammatory factors, is autoantigen; Immunity identification fragment is polypeptide or the dna fragmentation that immune system can be recognized as the dissident, and this fragment is positioned at SEQ18-23 and corresponding nucleotide sequence 40-42, is through selecting or chemical modification and remove the bacteriotoxin fragment of innate toxicity; The structure of described hybridization peptide is: one of aforesaid self inflammatory factor fragment is covalently bound each other with one of aforesaid immunity identification fragment.
The vaccine of the present invention's design is brand-new on chemical constitution: half in the employed hybridization peptide of this vaccine is the only very little fragment of amino terminal fragment of various chemotactic factors, have only 9-19 aminoacid size, only account for about 12-20% of the sequence of whole chemotactic factor, these small fragments all do not have the chemotactic factor activity; Second half of hybridization peptide adopts immunity identification fragment.
The result that the present invention obtains is beyond thought.Because under normal circumstances, because human body has the autoimmune toleration, body can not produce antibody to self material.Vaccine of the present invention destroyed this mechanism, makes body produce the antibody of anti-self chemokine protein.On the other hand, the antibody with one section little inducing peptide in the protein produces can not produce neutralization reaction with crude protein in general.This is because one section little peptide is a wire, induces the antibody of generation can only recognize the little peptide of wire.Usually, when the little peptide of this section as a whole proteic when part, the influence of other chemical group forms certain three dimensions conformation in the protein owing to being subjected to, and no longer is wire, thereby antibody can't be combined closely with crude protein.Hybridization peptide of the present invention inductive antibody but can play neutralization reaction with natural chemokine protein, this fragment that this explanation the present invention selects also is to exist with wire in whole protein structure.
In addition, because molecular weight product of the present invention is very little, so can adopt chemical synthesis process production, the product that adopts this method to produce need not purification, and technology is simple; Utilize another advantage of chemical synthesis process to be, except that can using natural L-aminoacid, also can use D-aminoacid to carry out sintetics.Because D-aminoacid is alpha-non-natural amino acid, can not be by proteasome degradation, so stable in vivo.And the product that biological engineering method is produced can only be to utilize natural L-aminoacid, and product subjects to decompose instability in vivo.By contrast, the present invention adopts the synthetic product of D-aminoacid to have clear superiority.
The present invention is by the big quantity research to the disease model of animal, establishes therapeutic scheme and be the main factor at the seizure of disease phase, rather than as most therapeutic scheme at the morbific factor, this is another unique aspect of the present invention.The chemotactic factor difference that different autoimmune diseases is related, and each disease often relates to multiple different chemotactic factor, is mainly concerned with IP-10 and MCP-1 etc. such as the allosome organ transplant rejection.And arthritis mainly is IL-8 and MCP-1.Research is also found, also changes to some extent at the chemotactic factor that the different phase that develops relates to a kind of disease.Another unique distinction of the present invention is to prepare mixed vaccine according to the kind of disease and development, can suppress multiple different chemotactic factor simultaneously, has both overcome the multiformity complexity of disease pathology, has kept the specificity to this disease again.Reach good effect probably, the effect that side effect is little.And only need do a clinical trial as mixed vaccine, need not do clinical trial respectively to every kind of composition, so the multiple medicine of exploitation is obviously much lower relatively for development cost; This vaccine is easy to use in addition, low price, and effect is lasting, is fit to very much the long-term autoimmune disease that relies on medicine.These advantage explanations mixed polypeptide vaccine of the present invention has very high clinical value probably.
The detailed description of invention
1, mixed polypeptide vaccine provided by the invention, its chemical constitution and composition characteristic are:
It is not of the same race by at least two kindsThe hybridization peptideMix and form.Can bring out the antibody of the chemotactic factor of anti-two or more differences of mammal secretion self in vivo.Wherein every kind of hybridization peptide all is by a kind ofBecomeChange the amino terminal fragment (a) of the factorWith a kind ofImmunity identification fragment (b)Covalently bound forming.Wherein the feature of the amino terminal small fragment of chemotactic factor is the activity that does not have chemotactic factor.Wherein the segmental feature of immunity identification is the toxicity that does not have original toxin.
Wherein, (a) the amino terminal fragment of chemotactic factor is the aminoterminal little fragments of peptides of various inflammation chemokine protein matter, and concrete sequence comprises that sequence 1 is to sequence 17 and corresponding nucleic acids sequence thereof.Specifically exemplify following constituent:
The amino terminal fragment of MCP-1 (sequence/SEQ1 and 24);
The amino terminal fragment of IP-10 (sequence/SEQ2 and 25);
The amino terminal fragment of ITAC (sequence/SEQ3 and 26);
The amino terminal fragment of MIG (sequence/SEQ4 and 27);
The amino terminal fragment of MCP-3 (sequence/SEQ row 5 and 28);
The amino terminal fragment of MCP-4 (sequence/SEQ6 and 29);
The amino terminal fragment of RANTES (sequence/SEQ7 and 30);
The amino terminal fragment of MIP-1aplpha (sequence/SEQ8 and 31);
IL-8 amino terminal fragment (sequence/SEQ9 and 32);
The amino terminal fragment of ENA-78 (sequence/SEQ10 and 33);
The amino terminal fragment of GRO-alpha (sequence 11 and 34);
The amino terminal fragment of SLC (sequence/SEQ12 and 35);
The amino terminal fragment of ELC (sequence/SEQ13 and 36);
And the sequence/SEQ13 of adnexa 1,14,15,16,17,37,38,39.
The amino terminal fragment of the chemotactic factor of wherein said hybridization peptide also has: with natural L-aminoacid or synthetic above-mentioned these sequences of non-natural D-aminoacid, the fragment of these sequences or analog; Its analog homology reaches 50%, 60%, 70% of above-mentioned sequence at least;
Above-mentioned these be the only very little amino terminal fragment of chemotactic factor, have only 9-19 aminoacid size, only account for about 12-20% of the sequence of whole chemotactic factor).The feature of these small fragments is the activity that do not have chemotactic factor (comprise with receptor binding capacity and inducing cell and move activity, referring under tabulate 1).Therefore, the little fragments of peptides of these non-activities can enhancing body yet to other proteinic immunity.The little fragments of peptides of these non-activities existsIn the hybridization peptideIt has been the effect of autoantigen.
Table 1: the result shows that chemotactic factor amino terminal fragment of the present invention does not have the functional activity of chemotactic factor.Functional examination comprises measures the binding ability of various chemotactic factor amino terminal fragments to chemokine receptors, and the ability of inducing target cell to move.The method that is adopted is summarized as follows:
(1) receptor binding assays: complete have active chemotactic factor (as MIP-1a, MCP-1 or the like) with isotope iodide 125 labelling in addition with to be determined.The determinand that the complete chemotactic factor of fixed amount labelling is commensurability is incubated with being subjected to somatic target cell.If determinand does not combine with this receptor, then isotope-labeled chemotactic factor and receptors bind amount remain unchanged.If determinand and receptors bind, then isotope-labeled chemotactic factor is subjected to the competition of determinand, must reduce with the receptors bind amount.
(2) induce target cell nigration method: chemotactic factor can induce its target cell to move towards the chemotactic factor direction.The effect of moving by the mensuration pair cell can detect function in the determinand.Method is to get a permeable membrane test board, and chemotactic factor or determinand are added under the test board in the face, then only adds target cell in the upper surface hole.Behind the insulation certain hour, the film that is subjected to chemotactic factor to induce target cell to move through between two holes enters down in the face.*
(table 1 is attached)
(b) described immunity identification fragment is that following sequence is formed:
The little peptide that one of the special fragment of tetanus toxin (sequence 18) has only 17 aminoacid to form; Dna fragmentation (sequence 19,20,21 of containing CpG; Such as the pCR2.1 that contains above-mentioned these polypeptide of sequence and nucleotide sequence thereof or pcDNA3.1 plasmid vector and viral vector);
The special fragment of cholera bacilli toxin (CT) (sequence 22),
The special fragment of the thermo-responsive toxin of Escherichia coli (LT) (sequence 23).
DescribedImmunity identification fragmentAlso has the fragment in above-mentioned these sequences; The analog of these sequences, its homology reach 50%, 60%, 70% of above-mentioned sequence at least; With natural L-aminoacid or synthetic these sequences of non-natural D-aminoacid replacement L-aminoacid, the fragment of these sequences or analog.
These fragments are cut DNA or the toxin that selects in antibacterial, and all through and selecting or chemical modification and remove original toxicity, but kept immunogenicity, be good immunological adjuvant.This part structure role in the hybridization peptide is to make immune system can discern exotic antigen (reference papers: 1.Kweon?MN，et?al，ANontoxic?Chimeric?Enterotoxin?Ad?juvant?Induces?Protective?Immunity?inBoth?Mucosal?and?Systemic?Compartments?with?Reduced?IgE?Antibodies.Infect?Dis?2002?Nov?1；186(9)：1261-9)。2。McCluskie，M.J.；et?al，Noveladjuvant?system，curr.Drug?Targets?Infect?Disorder，2001?1(3)：263-71)。The present invention especially selects the special fragment of this tetanus toxin wherein, because its molecular weight is little, is easy to chemosynthesis.
As the hybridization peptide of forming described mixed polypeptide vaccine composition, its another feature is: every kind of hybridization peptide is each other with covalent bond be formed by connecting the covalent bond of peptide bond or the definition of other chemistry (promptly by) by the amino terminal fragment of a specific aforesaid chemotactic factor one of (be among the sequence 1-17) and an aforementioned immunity identification fragment one of (be among the sequence 18-23).The direction that connects can be arbitrarily.Wherein hybridize the peptide structure and also comprise the nucleotide sequence (sequence 24-39 with 40-42 between be connected) of corresponding above-mentioned sequence.
The described mixed polypeptide vaccine of claim 1 is to be mixed with by two kinds or above hybridization peptide; Hybridization peptide of the same race is meant that their chemotactic factor amino terminal fragment is identical; Hybridization peptide not of the same race is meant that their chemotactic factor amino terminal fragment is inequality; Claim 10 is describedThe hybridization peptideStructure be: one of aforesaid self inflammatory factor fragment is covalently bound each other with one of aforesaid immunity identification fragment; Hybridization peptide of the same race is meant that they self inflammatory factor fragment is identical; Hybridization peptide not of the same race is meant that they self inflammatory factor fragment is inequality.
Change and modification to mixed polypeptide vaccine of the present invention:
Following is various rightThe present inventionThe change of mixed polypeptide vaccine and modification should be included within the present invention.Basically, if be to strengthen treatment or prophylactic effect in order to reach to the change of above-mentioned mixed polypeptide vaccine and modification; Or in order to improve product in vivo or external stability (to the defensive ability/resistance ability of body endoproteinase), such as with non-natural D-aminoacid replacement L-aminoacid; Or the modification that patent hybridization peptide is done in the synthetic back of pirate recordings, as changing saccharifying or phosphorylation composition, as long as the product that is derived by these variations remains with the principal character of this patent thing, that is their original antigenicities, both should be included within the present invention.
This modification comprises to be passed through to replace to the aminoacid of the aforementioned various hybridization peptides of the present invention or their nucleotide sequence, and increase or truncate come change, but the product that changes still keeps the principal character of patent thing.Can be replaced by isoleucine or propylhomoserin such as leucine, (or between basic amino acid, between the neutral amino acid, between the polar amino acid or between the nonpolar amino acid, between the aromatic amino acid) or the like can replace mutually between the acidic amino acid.It is generally acknowledged, identical more than 70% as long as the product after changing and the sequence of various inflammation polypeptide vaccines of the present invention have 50%, 60%, just should belong within the scope of the invention.
In addition,The present inventionThe aminoacid of aforementioned various hybridization peptides or their nucleotide sequence also can add other chemical compound and keep on its pendant chemical groupsThe present inventionThe amino acid whose principal character of aforementioned various hybridization peptides as alkali, thereby increases its solubility; Or isotope or fluorescence or the like, reaching in the body or external tracer action, thereby be applied to diagnosis to target cell.
In addition,The present inventionThe hybridization peptide of mixed polypeptide vaccine or their nucleotide sequence also can be hybridized with the protein of other function or their nucleotide sequence again.Than hybridizing again, to reach the effect that strengthens therapeutic effect with the tumor necrosis factor fragment.These all are the methods of widespread.
The preparation method of mixed polypeptide vaccine of the present invention:
The first step: produce the aforesaid various hybridization peptides of preparation.Can adopt following four kinds of methods to produce the various hybridization peptides of preparation: to utilize chemical synthesis process production preparation; Utilize gene recombination technology to produce preparation; Produce the method for preparing the hybridization peptide that is connected with the dna fragmentation that contains CpG by chemotactic factor amino terminal polypeptide fragment; The method of excretory hybridization peptide prod separation and purification will be produced.The basic fundamental that these methods can be used for a small amount of preparation or produce in batches.
In second step, be prepared into the mixed polypeptide vaccine According to kinds of Diseases and involved chemotactic factor of PD stage, select two or more suitable hybridization peptides to mix at these factors, again this mixture is prepared into the mixed polypeptide vaccine.
The detailed description of above-mentioned preparation method:
The first step: produce the aforesaid various hybridization peptides of preparation:
A,Utilize chemical synthesis process production to prepare various hybridization peptides: the hybridization chemistry of peptides is synthetic to utilize the peptide solid phase synthesis technique, both can utilize the polypeptide automatic synthesizer to synthesize, but synthetic again.Standard method such as Bodansky, M, Principles of peptide synthesis, springer Verlag, Berlin (1993) and Grant, G.A. (ed), Synthetic peptides:A user ' s guide, W.H.Freeman and Company, New York (1992). for example, concrete grammar of the present invention is to utilize the polypeptide automatic synthesizer next synthetic: synthetic carboxyl terminal from the hybridization peptide.According to the sequence of the hybridization peptide of selecting design will be with the aminoacid of good this sequence carboxyl terminal of resin coupling as initial, side-chain radical is connected up by peptide bond one by one by 20 seed amino acids that chemical group (as t-BOC or f-MOC group) is protected.In order to ensure synthetic efficient; behind each aminoacid Connection Step; monitor the success rate that peptide bond connects; monitoring method such as J.M.Stewart and J.D.Young (ed); Solid Phase Peptide Synthesis; Pierce Chemical Company (1984). after whole peptide sequence connection was finished, each blocking group was removed with chemical method (as fluohydric acid gas) the most at last.Product through as high pressure liquid chromatograph or anti-phase layer suction post layer adsorption technology, or affinity layer adsorption technology purification in addition.But because hybridization peptide molecular weight of the present invention is little, reactions steps is few, the productive rate height, can reach generally speaking 85 percent or more than, so need not purification.In addition, synthetic little peptide also need not " fold (folding) " to form three grades of space structures, can directly use (embodiment 1) as product.
Owing to be chemosynthesis,, can use non-natural D-aminoacid to synthesize the hybridization peptide again so both can use L-aminoacid.The synthetic hybridization peptide of D-aminoacid is not destroyed by protease, and is stable in the body.In addition, some other chemical group, as biotin (Biotin), fluorophor etc. also can be synthesized in the hybridization peptide sequence and go, with as detection or diagnostic preparation.
B, utilize gene recombination technology to produce the various hybridization peptides of preparation: to utilize gene recombination technology can produce the various hybridization peptides of preparation.At first design 5 ' and 3 ' primer, add single restriction restriction endonuclease point of contact sequence on the head of primer, so that be connected with carrier DNA at each sequence according to sequence 1-23 in the adnexa 1.In addition, 5 ' primer also must add initial signal, and 3 ' primer also must add termination signal.Utilize these special primers, the aminoterminal genetic fragment cDNA of various chemotactic factors and the segmental genetic fragment cDNA of aforesaid immunity identification can be cloned among the RNA by the RNA of people's cell and antibacterial respectively by the RT-PCR method.Utilize clone technology that the aminoterminal genetic fragment cDNA of each chemotactic factor is connected by the restriction restriction enzyme site with immunity identification fragment sequence again, form the hybrid gene of expressing the hybridization peptide.
In addition, the invention provides to expressing the necessary carrier of above-mentioned hybridization peptide.The carrier that carrier has bacterial plasmid vector or eukaryotic cell to use is such as example pCR2.1 shown in the embodiment 2 and pCDNA3.1 plasmid vector.Make things convenient for the impedance gene of carrier threading at different antibiotics, anti-ampicilin that analogy utilizes and the gene of anti-kanamycin herein in order to screen.In order to increase output, added enhancer in the carrier.Above-mentioned cDNA is cloned in the carrier.
This patent also provides the bacterial strain that has various hybridization peptide gene carriers.The carrier that has various hybridization peptide genes is expressed propagation through transfecting host (such as antibacterial).Use escherichia coli DH α 5 as the host such as selecting in the example 2.Plamid vector transfection DH α 5 with various hybridization peptide genes.Obtain the hybridization peptide of a large amount of expression then by the bacterial strain of a large amount of these transfections of breeding.Because what this method was produced is to contain the bacterial mixture of hybridizing peptide, product must purification (concrete grammar as follows).On the other hand, if the hybridization peptide molecular weight is big, such as containing the segmental hybridization peptide of cholera bacilli toxin (CT), then product must through " folding (folding) ' ' to form three grades of space structures.The general in this way method in common of its method, as J.M.Stewart and J.D.Young (ed), Solid Phase Peptide Synthesis, PierceChemical Company (1984) is described.
C, production prepare the method for the hybridization peptide that is connected with the dna fragmentation that contains CpG by chemotactic factor amino terminal polypeptide fragment: the hybridization peptide of this method preparation is to be formed by connecting by little fragments of peptides of chemotactic factor amino terminal and the dna fragmentation that contains CpG: at first, utilize above-mentioned chemical synthesis or gene recombination technology to prepare earlier the little peptide of various chemotactic factor amino terminals respectively.On the other hand, utilize the synthetic dna fragmentation (sequence 19-21) that contains CpG of chemical synthesis process (as utilizing common automatic dna synthesizer), or isolate the dna fragmentation that contains CpG from the DNA of antibacterial.Utilize chemical reaction method that they are covalently bound again.Covalently bound method has multiple, analogy is here with reference to Hearn J.Cho et al, ImmunostimulatoryDNA-based vaccines induced cytotoxic lymphocyte activity by a T-helpercell-independent mechanisms, Nature Biotech.2000,18:509-514. simple description the: chemotactic factor amino terminal polypeptide fragment is activated by reacting with compound S ulfo-SMCC, simultaneously, will be connected to the segmental phosphate group of CpG-DNA with disulphide.The two is mixed in varing proportions, it is reacted to each other than this, thereby prepare the hybridization peptide that is formed by connecting by little fragments of peptides of chemotactic factor amino terminal and the dna fragmentation that contains CpG.
D, the present invention further provides the method that how will produce the separation and purification of excretory hybridization peptide prod: what adopt is the partition method of affinity chromatography herein.Affinity chromatography can use the anti-segmental antibody of aforesaid chemotactic factor amino terminal.Method is at first institute to be adopted to contain the segmental hybridization peptide of this chemotactic factor amino terminal, such as coming immune animal (as rabbit) with MCP1 amino terminal hybridization peptide-1 (N-MCP1-TT).Through booster immunization several times, with the rabbit blood-letting.Affinity chromatographic column with little peptide of MCP1 amino terminal or a-protein preparation is purified the antibody in the blood.These antibody are the antibody of the anti-MCP1 amino terminal hybridization of rabbit peptide-1.These isolated antibody are covalently bound to the resin as carrier support with conventional chemical reaction method, be prepared into affinity chromatographic column.This affinity chromatographic column can be used to separation and purification MCP1 amino terminal hybridization peptide.Method is will produce the impure hybridization peptide product of preparation (for example aforesaid utilize the bacterioprotein mixture that containing of bacterial expression hybridize peptide) to be added on the above-mentioned affinity chromatographic column.Since hybridization peptide contain MCP1 amino terminal fragment, thereby can with the antibody specific bond on the affinity chromatographic column, thereby separate with other impurity, reach the purpose of purification.
In second step, utilize the hybridization peptide to prepare the method for mixed polypeptide vaccine:
Another unique distinction of the present invention is to prepare mixed vaccine according to the kind of disease and development.At first add with the ELISA method according to kinds of Diseases and PD stage and test the chemotactic factor of confirming that such disease pathology is involved, such as arthritis, it mainly is that IL-8 and MCP-1 express and improve, and psoriasis mainly to be IP10 and IL-8 express improves.Research is also found, also changes to some extent at the chemotactic factor that the different phase that develops relates to a kind of disease.Select two or more suitable hybridization peptides to mix at these chemotactic factors, analogy is adopted arthritis and is mixed with the segmental hybridization peptide of IL-8 amino terminal by the segmental hybridization peptide of MCP-1 amino terminal.After mixing, help the chemical compound of efficacy of vaccines to become mixed vaccine according to method co-production commonly used with adjuvant commonly used (as aluminium hydroxide, mineral oil) or other.Such benefit is that vaccine had both overcome the multiformity complexity of disease pathology, had kept the specificity to this disease again.
1, the technical characterictic of invention mixed polypeptide vaccine and evaluation thereof:
The technical characterictic of mixed polypeptide vaccine of the present invention
The feature of mixed polypeptide vaccine of the present invention is with regard to its constituent, the feature of promptly aforementioned various hybridization peptides.Have two kinds of features: chemical structure characteristic and antigen property.
(1) chemical structure characteristic: aforementioned various hybridization peptides all are to be formed by connecting by two parts: promptly the aminoterminal little fragments of peptides of aforesaid chemotactic factor is discerned the hybridization peptide that the fragment binding is become with immunity.Wherein every kind of hybridization peptide all is by a kind ofThe amino terminal fragment of chemotactic factor (Specifically see sequence 1 to sequence 17, and corresponding nucleic acids sequence 24-39) with a kind ofImmunity identification fragment (Specifically see sequence 18-23, and shown in the corresponding nucleic acids sequence 40-42) covalently bound forming.So the sequence of every kind of hybridization peptide must be one of combinations thereof.Concrete sequence is exemplified below (being not limited to):
A. MCP1 amino terminal in people source is hybridized peptide-1 (N-MCP1-TT):
QPDAINAPVTC?QYIKANSKFIGITELKK
B. Mus source N-MCP1-hybridization peptide-2:(N-MCP1-CpG-DNA):
QPDAVNAPLTCCYSFT...tgactgtgaacgttcggatga..
C. IP10 amino terminal in people source is hybridized peptide-1 (N-IP10-TT):
VPLSRTVRCTCISIQYIKANSKFIGITELKK
D. IL-8 amino terminal in people source is hybridized peptide-1 (N-IL8-TT):
SAKELRCQCIKTQYIKANSKFIGITELKK
E. Mus source MCP-1 (JE) amino terminal is hybridized peptide-1 (N-MCP1-TT):
QPDAVNAPLTCCYSFTQYIKANSKFIGITELKK
F. IP10 amino terminal in Mus source is hybridized peptide-1 (N-IP10-TT):
IPLARTVRCNCIHIQYIKANSKFIGITELK
G. MIP2 amino terminal in Mus source is hybridized peptide-1 (N-MIP2-TT):
AVVASELRCQCLKTQYIKANSKFIGITELKK
(2) antigen property: the present invention forms the antibody that the hybridization peptide of mixed polypeptide vaccine can bring out with it as antigen specific association reaction.Specifically, with hybridization peptide A as the antibody that antigen-immunized animal obtained, can with hybridization peptide A specific bond, produce positive reaction.This is because antigen has specificity: it is mutually identical with relation between its antibody, mutually at, be single-minded.
Evaluation to mixed polypeptide vaccine feature of the present invention:
Evaluation to the feature of mixed polypeptide vaccine of the present invention is exactly to its constituent, the evaluation of the feature of promptly aforementioned various hybridization peptides.Aforementioned various hybridization peptide all is to be formed by connecting by two parts: i.e. fragment is discerned in the aminoterminal little fragments of peptides of aforesaid chemotactic factor and immunity.Because the aminoterminal little fragments of peptides of aforesaid chemotactic factor does not all have the chemotactic factor activity, aforesaid immunity identification fragment no longer has toxicity, identifies so can't check by activity, can only pass through its chemical constitution, that is its sequence is identified.
The evaluation of its chemical constitution can promptly, 1. adopt mass spectrograph that the sequence of the hybridization peptide in the mixed polypeptide vaccine is identified by following two approach; 2. by its special antigenicity being identified at the antibody of various hybridization peptide preparations.
Concrete method of testing: 1. 2.
At first the component of mixed polypeptide vaccine production thing is separated.Separating has method not of the same race, and such as utilizing the polypropylene gel electrophoresis to separate, different components rest on the diverse location of gel, form different bands.Each band is downcut respectively, and middle component extracts in will being with then.Also can utilize chromatography in addition,, separate as high pressure liquid chromatography.Different components is collected in the different eluents.
1. determine the aminoacid or the nucleotide sequence of hybridization peptide.
The sequence of above-mentioned isolating each component is measured: the sequence of polypeptide utilizes mass spectrograph to measure.Assay method as standard method commonly used; If the hybridization peptide contains DNA, then DNA sequence is measured according to conventional method with the nucleic acid automatic sequencer.
2. determine the antigenicity of hybridization peptide.
Measure above-mentioned isolating each component and whether contain the specific antigenicity of hybridizing peptide diverse ways is arranged, commonly used as integrated enzyme reaction method and West Block method.Two kinds all is the method that is in daily use.
Must prepare specific antibody before measuring at various hybridization peptides.Preparation method is as follows: at first with the hybridization peptide that is adopted, come immune animal (as rabbit) respectively such as personnel selection source MCP1 amino terminal hybridization peptide-1 (N-MCP1-TT) and people source RANTES amino terminal hybridization peptide-1 (N-RANTES-TT) for example.Through booster immunization several times, with the rabbit blood-letting.Be prepared into two affinity chromatographic columns respectively with MCP-1 amino terminal fragment or RANTES amino terminal fragment.Utilize affinity chromatography that the antibody in the blood is purified respectively.These antibody of Ti Chuning are the antibody of the anti-people of rabbit source MCP1 amino terminal hybridization peptide-1 (N-MCP1-TT) and the antibody of the anti-people of rabbit source RANTES amino terminal hybridization peptide-1 (N-RANTES-TT) thus.Make the antibody of anti-other hybridization peptide in accordance with the law.
Next step identifies the hybridization peptide specific antigenicity of above-mentioned isolating each component with the integrated enzyme reaction method.Method is as follows: with the ingredient of known anti-hybridization peptide X, such as the antibody of the anti-people of rabbit source MCP1 amino terminal hybridization peptide-1 (N-MCP1-TT), insulation closely is adsorbed onto on the cellular-plastic panels by spending the night.Above-mentioned isolating each component is diluted to variable concentrations, is added to afterwards on this plastic plate.Flush away material unconjugated repeatedly with it.Add that afterwards enzyme mark second antibody (such as the goat anti-rabbit antibody with the hydrogen peroxide enzyme labelling) develops the color.Whether same manner is measured this component and is reacted with the antibody of the anti-people of rabbit source RANTES amino terminal hybridization peptide-1 (N-RANTES-TT).If it is positive that first reaction is colour developing, second reaction is negative, illustrate to contain MCP1 amino terminal hybridization peptide in people source in this mixed polypeptide vaccine, and nobody source RANTES amino terminal hybridization peptide.
2, the invention provides the purposes of aforesaid mixed polypeptide vaccine:
The described mixed polypeptide vaccine of the one, can be applicable to prepare the vaccine for the treatment of or preventing all kinds of autoimmune diseases and organ-graft refection's relevant disease; Wherein, described autoimmune disease comprises arthritis, rheumatism, dry syndrome, lupus erythematosus, colitis, psoriasis, multiple hard flesh disease, scleroderma; Described organ-graft refection's relevant disease refers to repulsion and the graft versus host disease that organ transplantation causes.
The dosage that wherein said mixed polypeptide uses when making vaccine: its using dosage is 10 micrograms-500 milligram/people;
The dosage form of the vaccine that wherein said mixed polypeptide is made is made up of following: the absorption type of injection type, peroral dosage form and skin, nasal cavity, genitals, intestinal, the injection site of the vaccine that wherein said mixed polypeptide is made can be at Intradermal, subcutaneous or intramuscular.
The purposes of the aforesaid mixed polypeptide vaccine of the 2nd, can be used for preparing diagnostic reagent.
3, beneficial effect of the present invention
The present invention designs and makes the mixed polypeptide vaccine of a series of treatment autoimmune disease.For therapeutic effect and the mechanism of testing this vaccine, we utilize mouse disease model and the chemotactic factor vaccine (seeing embodiment for details) of himself to measure.The result shows that this class vaccine can suppress the invade profit of leukocyte to the inflammation tissue, reaches the effect that reduces recurrence and treatment autoimmune disease.This is likely because of vaccine has destroyed in the body toleration to self chemotactic factor, thereby brings out and produce corresponding antibody in the body.These antibody can neutralize when inflammation in the body self excretory inflammation chemokine protein matter, thereby play therapeutic effect.
Result of the test shows that not only preparation technology is simple for these mixed polypeptide vaccine bodies, and interior curative effect is permanent. thereby these mixed polypeptide vaccines make those patients that rely on medicines throughout one's life can suppress morbidity probably, and don't with take medicine everyday.This will bring great convenience to patient, and save a lot of expenses.
This vaccine is convenient in addition uses, low price, and effect is lasting, is fit to very much the autoimmune disease that general medicine is difficult to disease controlling.These advantages show that mixed vaccine of the present invention has very high clinical value probably.
The vaccine of the present invention's design is brand-new on chemical constitution.In the employed hybridization peptide of this vaccine half is the only very little fragment (generally have only 9-19 aminoacid size, and the about 70-111 of a whole chemotactic factor aminoacid) of various chemotactic factors, and second half of hybridization peptide adopted immune identification fragment.The result that we obtain is beyond thought.Because under the normal condition, animal body can not produce antibody to self material, and our vaccine of invention has destroyed this mechanism, makes body produce the antibody of anti-self chemokine protein.On the other hand, the antibody with one section little inducing peptide in the protein produces can not produce neutralization reaction with crude protein in general.This is because one section little peptide is a wire, induces the antibody of generation can only recognize the little peptide of wire.Usually, when the little peptide of this section as a whole proteic when part, the influence of other chemical group forms certain three dimensions conformation in the protein owing to being subjected to, and no longer is wire, thereby antibody can't be combined closely.And our hybridization peptide inductive antibody can play neutralization reaction with natural chemokine protein.This illustrates that this fragment that we select also is to exist with wire in whole protein structure.
On the other hand, we are by the big quantity research to the disease model of animal, establish therapeutic scheme and be the main factor at the seizure of disease phase, rather than as most new drug researches only at the morbific factor.This is another unique aspect of the present invention.In addition, the chemotactic factor difference that different autoimmune diseases is related, and also each disease often relates to multiple different chemotactic factor.Analogy allosome organ transplant rejection is mainly concerned with IP-10 and MCP-1 etc.And arthritis mainly is IL-8 and MCP-1.Research is also found, also changes to some extent at the chemotactic factor that the different phase that develops relates to a kind of disease.Another unique distinction of the present invention is to prepare mixed vaccine according to the kind of disease and development, can suppress multiple different chemotactic factor simultaneously, has both overcome the multiformity complexity of disease pathology, has kept the specificity to this disease again.Curative effect is more far better than a kind of chemotactic factor of simple inhibition.And only need do a clinical trial as mixed vaccine, need not do clinical trial respectively to every kind of composition.So development cost are low.
In addition, because our molecular weight product of invention is very little, adopt the synthetic production of chemical method, product need not purification, and technology is simple.Because the chemical method sintetics is used in our invention, so except natural L-aminoacid, we also use D-aminoacid to come sintetics.Because D-aminoacid is alpha-non-natural amino acid, can not be by proteasome degradation, so stable in vivo.And the product that they use biological engineering method to produce can only be to utilize natural L-aminoacid, instability.
Description of drawings
The present invention is further described below in conjunction with drawings and Examples.
Fig. 1 a is in the arthritis process of the test of the present invention, the relevant pathological section result who brings out leg joint behind the arthritic mice: all kinds of leukocyte percentage ratio sketch maps of invading the profit joint tissue.
Fig. 1 b is in the arthritis process of the test of the present invention, chemokine gene expression sketch map in the relevant pathological tissue that brings out leg joint behind the arthritic mice.
Fig. 2 a is in the arthritis process of the test of the present invention, the sketch map that the relevant mice back leg arthroncus of accepting the mixed polypeptide vaccine immunity treatment of single vaccine (N-MIP-TT or N-MCP1-TT) or above two kinds of compositions changes.Wherein matched group is not treatment group.
Fig. 2 b is in the arthritis process of the test of the present invention, and the relevant mixed polypeptide vaccine immunity of accepting single vaccine (N-MIP-TT or N-MCP1-TT) or above two kinds of compositions is treated the sketch map of the joint pathology extent of damage.Wherein matched group is not treatment group.
Fig. 3 is in the arthritis process of the test of the present invention, the relevant long-term efficacy of accepting the mixed polypeptide vaccine immunity treatment of single vaccine (N-MIP-TT or N-MCP1-TT) or above two kinds of compositions.Be depicted as the overall performane sketch map of 2 to 3 months posterior joint pathological lesion degree of receiving treatment.Wherein matched group is not treatment group.
Fig. 4 re-uses the mixed polypeptide vaccine therapy effect that N-MIP-TT and N-MCP1-TT form in the arthritis process of the test of the present invention after arthritis forms, the sketch map that its mice back leg arthroncus changes.Wherein matched group is not treatment group, and the group that utilizes the little peptide of immunity identification fragment to do non-specific therapy.
Fig. 5 is in the arthritis process of the test of the present invention, the sketch map that the relevant mice back leg arthroncus of accepting the mixed polypeptide vaccine immunity treatment of single vaccine (N-MIP-DNA or N-MCP1-DNA) or above two kinds of compositions changes.Wherein matched group is not treatment group.
Fig. 6 accepts the mice of the mixed polypeptide vaccine immunity treatment of single vaccine (N-MIP-TT or N-MCP1-TT) or above two kinds of compositions, to anti-MCP-1 and the anti-MIP-2 detection of antibodies result who produces in its blood in the arthritis process of the test of the present invention.Be depicted as the relative amount of above-mentioned two kinds of antibody, represent with the antiserum dilution factor.This antibody content height of the high expression of dilution factor.
Fig. 7 a is in the process of the test of dry syndrome of the present invention, the pathological section result of the dry syndrome of the relevant spontaneous generation of mice: all kinds of leukocyte percentage ratio sketch maps of invading profit salivary organization.
Fig. 7 b is in the process of the test of dry syndrome of the present invention, the chemokine gene expression sketch map in the pathological tissue (salivary gland) of the dry syndrome of the relevant spontaneous generation of mice.
Fig. 8 is in the process of the test of dry syndrome of the present invention, the relevant mice of accepting the mixed polypeptide vaccine therapy of N-MIP-TT and N-IP10-TT composition, the salivary gland pathology index sketch map of its dry syndrome.Wherein matched group is not treatment group.
Fig. 9 is in the process of the test of dry syndrome of the present invention, the relevant mice of accepting the mixed polypeptide vaccine therapy of N-MIP2-TT and N-IP10-TT composition, anti-IP-10 that produces in its blood and anti-MIP-2 detection of antibodies result.Be depicted as the relative amount of above-mentioned two kinds of antibody, represent with the antiserum dilution factor.This antibody content height of the high expression of dilution factor.
Figure 10 is in the LE test process of the present invention, about accepting the mice of the mixed polypeptide vaccine therapy that N-MIP2-TT and N-IP10-TT form, and the mice of accepting the mixed polypeptide vaccine therapy of N-MIP2-TT and N-MCP1-TT composition, its kidney cell is invaded profit pathology index sketch map.Wherein matched group is not treatment group.
The specific embodiment
1, about the composition and the preparation of mixed polypeptide vaccine of the present invention, show as follows as an example.The concrete method that adopts:
The first step: the hybridization peptide of mixed polypeptide vaccine is formed in preparation.Adopt two kinds of method preparations to form the hybridization peptide of mixed polypeptide vaccine altogether at this: (1) utilizes chemical method to synthesize (example 1); (2) utilize gene recombination method preparation (example 2).Concrete grammar is seen following embodiment 1 and embodiment 2 for example; (3) product of separation and purification production is the partition method (embodiment 2) of affinity chromatography shown herein.
Second step: utilize the hybridization peptide to prepare the mixed polypeptide vaccine.Mixed vaccine is to prepare according to the kind of disease herein.At first add with the integrated enzyme reaction method and test the chemotactic factor of confirming that such disease pathology is involved according to kinds of Diseases and PD stage, it mainly is that MIP-2 and MCP-1 express and improve such as the arthritis (embodiment 3-6) of mice, and dry syndrome mainly to be IP10 and MIP-2 express improves.
Select two or more suitable hybridization peptides to mix at every kind of related inflammation chemotactic factor of disease, suppose that above-mentioned arthritis employing is mixed with the segmental hybridization peptide of MIP2 amino terminal by the segmental hybridization peptide of MCP-1 amino terminal.After mixing, become mixed polypeptide vaccine (embodiment 3-10) according to method co-production commonly used with adjuvant Freund's complete adjuvant commonly used.
Embodiment:
Embodiment 1: the chemosynthesis of Mus MCP1 amino terminal hybridization peptide (called after N-MCP1-TT) since selected be the curative effect that model comes test vaccine with the mice, vaccine must be made of the aminoterminal hybridization peptide of mice chemotactic factor, with as autoantigen.Now the N-MCP1-TT with Mus is an example, and its chemical synthesis process is described.Concrete sequence is as follows:
QPDAVNAPLTCCYSFTQYIKANSKFIGITELKK
It is to be formed by connecting by peptide bond by Mus MCP1 amino terminal fragment and the special fragment of tetanus toxin, with it called after N-MCP1-TT.
Description was partly done in the standard method of chemosynthesis preparation method in front.For instance, the concrete grammar that we adopted is to utilize the polypeptide automatic synthesizer next synthetic.Sequence according to the hybridization peptide of selecting; with with the amino acid lysine of good this sequence carboxyl terminal of resin coupling as initial, 20 seed amino acids that side-chain radical has been protected by chemical group t-BOC group are according to being begun to connect up by peptide bond one by one by carboxyl terminal.In order to ensure synthetic efficient, behind each aminoacid Connection Step, we will monitor the success rate that peptide chain connects.Each blocking group is removed with hydrogen fluoride reaction the most at last.Through high-pressure liquid chromatography, the purity of product is not purified just to be reached approximately 85 percent, so do not need separation and purification, it is also synthetic by Same Way to be that simple cleaning can directly be used for preparing other hybridization peptide of vaccine.
Following is the sequence (example, but be not limited to) of some synthetic hybridization peptides:
People source N-MCP1-TT:QPDAINAPVTCCYNFTQYIKANSKFIGITELKK
People source N-IP10-TT:VPLSRTVRCTCISISQYIKANSKFIGITELKK
People source N-IL8-TT:SAKELRCQCIKTQYIKANSKFIGITELKK
Mus source N-MCP1-TT:QPDAVNAPLTCCYSFTQYIKANSKFIGITELKK
Mus source N-IP10-TT:IPLARTVRCNCIHIDDQYIKANSKFIGITELKK
Mus source N-MIP2-TT:AVVASELRCQCLKTQYIKANSKFIGITELKK
The gene recombinaton of the amino terminal fragment hybridization peptide (N-IL8-DNA) of embodiment 2:IL-8 reaches in colibacillary expression
The gene of the amino terminal fragment of chemotactic factor IL-8 (adnexa 1, sequence 8) obtains from people's mononuclear cell mRNA storehouse by the RT-PCR method.The 5 '-primer of Shi Yonging is 5 '-ttgctagctccaccatgacttccaagctgg-3 ' for this reason; 3 '-primer is: 5 '-tatagcggccgcggagtatgtctttat-3 '.The cDNA of this gene is cloned into earlier in the PCR2.1 plasmid vector.Again IL-8 amino terminal fragment gene (called after N-IL8) is downcut with restricted enzyme NheI and HindIII, change and be cloned into (this carrier contains the dna fragmentation of CpG, can express in mammalian cell again) in the pCDNA3.1 expression plasmid carrier.The plasmid pcDNA3.1 transfection Escherichia coli DH α 5 that will have gene, and in the culture fluid that contains Ampicillin antibiotics, cultivate these antibacterials in a large number.Utilize general conventional method the plasmid that has hybridization peptide gene can be separated and prepare, be directly used in the preparation vaccine.
Except said method, also the method that can adopt chemosynthesis to combine with thorugh biologic engineering method prepares hybridization peptide, for example N-IL8-DNA.Concrete grammar partly is described in aforementioned preparation method.Now be briefly described as follows: the hybridization peptide of this method preparation is by the little fragments of peptides of chemotactic factor amino terminal chemosynthesis or that the clone gets well, as the amino terminal fragment (sequence 8) of IL-8, is formed by connecting with the dna fragmentation that contains CpG.Method is to utilize aforementioned chemical synthesis or gene recombination technology to prepare earlier the little peptide of IL-8 amino terminal respectively.Wherein the how little peptide of IL-8 amino terminal of gene recombination technology production is mixed in together with many compositions of antibacterial, thereby must be through separation and purification.Specifically be affinity chromatography as used herein: at first prepare the little peptide antibody of the anti-IL-8 amino terminal of rabbit (method of utilizing the part of preparation method as described above to be introduced).These antibody are covalently bound to as on carrier support and the Sepharose-4B that activated with conventional chemical reaction method, be prepared into affinity chromatographic column.This affinity chromatographic column can be used to the little peptide of separation and purification IL-8 amino terminal.Method is that the mixture that contains the little peptide of IL8 amino terminal that will produce preparation is added on the above-mentioned affinity chromatographic column.Since the little peptide of IL-8 amino terminal can with the antibody specific bond on the affinity chromatographic column, thereby separate with other impurity, reach the purpose of purification.
On the other hand, utilize the synthetic dna fragmentation (sequence 19-21) that contains CpG of chemical synthesis process (as utilizing common automatic dna synthesizer), or isolate the dna fragmentation that contains CpG from the DNA of antibacterial.Utilize chemical reaction method that the little peptide of chemotactic factor amino terminal is covalently bound with the DNA that contains CpG again, be prepared into the hybridization peptide.
2, the application of mixed polypeptide vaccine of the present invention:
The present invention designs and makes the mixed polypeptide vaccine of a series of treatment autoimmune disease.For therapeutic effect and the mechanism of testing this vaccine, we utilize mouse disease model: arthritis model, dry syndrome model and lupus erythematosus model are measured its curative effect.We have prepared the mixed polypeptide vaccine at different mice self chemotactic factor, and it is used on these disease models.The result shows that this class vaccine can suppress the invade profit of leukocyte to the inflammation tissue, reaches the effect that reduces recurrence and treatment autoimmune disease.This is likely because vaccine has destroyed in the body toleration to self chemotactic factor, thereby brings out and produce corresponding antibody in the body.These antibody can neutralize when inflammation in the body self excretory inflammation chemokine protein matter, thereby play therapeutic effect.Illustrate as arthritis, because its stage of attack chemotactic factor MCP-1, the level of MIP-2 (quite people IL-8) increases obviously that (embodiment 3,4,5), thus the treatment vaccine adopt and to contain Mus MCP1 amino terminal hybridization peptide (N-MCP1-TT) and two kinds of hybridization peptides making of Mus MIP2 amino terminal hybridization peptide (N-MIP2-TT, quite people's N-IL8-TT) mixed vaccine (embodiment 3,4,5).Again such as period of expansion IP-10 in dry syndrome and systemic lupus erythematosus (sle), MCP-1 and MIP-2 obviously increase, then adopt and contain three kinds of Mus MCP1 amino terminal hybridization peptide (N-MCP1-TT) and Mus MIP2 amino terminal hybridization peptide (N-MIP2-TT) and Mus IP10 amino terminal hybridization peptides (N-IP10-TT) and hybridize peptide making mixed vaccine (embodiment 8,9,10).The therapeutic effect of mixed vaccine is more remarkable than single vaccine generally speaking.Embodiment 4 result of the tests show that also curative effect is permanent in vivo for these mixed polypeptide vaccines. see embodiment 3,4 for details, 6,7,8,9,10.These test also offered high-quality service mixed polypeptide vaccine using method and dosage.
This vaccine is convenient in a word uses, and effect is obvious, and effect is lasting, is fit to the autoimmune disease that general medicine is difficult to disease controlling.These advantages show that the mixed polypeptide vaccine has very high clinical value.
Embodiment:
Below all in vivo tests all be to do with the MRL-1pr mice.This mice is owing to be FAS ligand gene defective, thereby spontaneously produces a series of autoimmune disease, comprising arthritis, and dry syndrome and lupus erythematosus.Be autoantigen, the sequence of the Mus that following employed relevant chemotactic factor amino terminal sequence all is meant.
Embodiment 3: Mus MCP1 amino terminal hybridization peptide (N-MCP1-TT) and Mus MIP2 amino terminal hybridization peptide (N-MIP2-TT) mixed polypeptide vaccine suppress mouse arthritis.The MRL-lpr mice makes the spontaneous generation arthritis in 24 all left and right sides Mus.By giving fully not formula adjuvant of mice intradermal injection, can induce arthritis (about 14 weeks) generation ahead of time, and symptom is obvious, is beneficial to observation of curative effect.Fig. 1 a shows, during the arthritis outbreak, invading a large amount of mononuclear cell of profit and neutrophilic granulocyte in the articular cavity (utilizes immuning dyeing method to identify, that is utilize the anti-antigenic antibody of various leukocyte surfaces to invading the leukocyte dyeing of profit, all kinds of leukocyte are counted respectively, its numeral accounts for the percentage ratio of total intrusion cell again).Utilize limited PCR method (only doing 20-30 circulation when being PCR amplifies) research chemokine gene to express.Express the multiple (Fig. 1 b) that improves by expressing to compare to provide with the normal structure chemokine gene.The result shows that the inflammation chemotactic factor of main expression is MCP-1 and MIP-2.
At above-mentioned discovery, we have prepared the mixed vaccine of being made up of segmental hybridization peptide of MCP-1 amino terminal (N-MCP-TT) and the segmental hybridization peptide of MIP2 amino terminal (N-MIP-TT).Concrete grammar is as follows: Mus N-MCP1-TT and these two kinds hybridization peptides of Mus N-MIP2-TT hybridization peptide (sequence is seen before to state and implemented embodiment 1) are pressed 1 to 1 mixed.After mixing,, become mixed vaccine according to method co-production commonly used with adjuvant commonly used, Freund's complete adjuvant.Ultimate density: two kinds of hybridization peptides respectively are 0.5 mg/ml.For the effect of mixed polypeptide vaccine relatively, only also prepared by a kind of in above-mentioned and hybridized the single vaccine that peptide is made with adjuvant, final concentration is 1 mg/ml.
In order to study curative effect, the MRL mice in four group of 13 week, every group of 12-15 only, subcutaneous inject respectively 0.5 milliliter by N-MCP-TT, or the single vaccine made of N-MIP-TT (100 micrograms/only), or two kinds of mixed polypeptide vaccines that all have.A matched group injection solvent and adjuvant.Per two weeks are partly measured duplicate injection once, totally 3 times.In order to induce arthritis, after injection for the second time, give fully not formula adjuvant of all mice intradermal injections.Fig. 2 a shows that not treatment group is the arthroncus of not treatment group after inducing about 12-15 days, inflammation.Bone slice shows that a large amount of leukocyte invades articular cavity and joint subcutaneous connective tissue after 25 days, causes the permanent lesion (Fig. 2 b) of cartilage and osseous tissue.Make moderate progress though treat the symptom and the pathology of group through single vaccine immunity, symptom is still obvious, and its arthritis of mixed polypeptide vaccine processed group remains dormant substantially.The result shows that N-MCP1-TT and N-MIP2-TT mixed vaccine can stop arthritic recurrence, and effect is more remarkable than single vaccine.
Embodiment 4.Mus MCP1 amino terminal hybridization peptide (N-MCP1-TT) and Mus MIP2 amino terminal hybridization peptide (N-MIP2-TT) mixed polypeptide vaccine suppress the long-term efficacy of mouse arthritis.
Present embodiment is all to induce arthritis and immunization therapy mice according to the method for embodiment 3.Afterwards, without any further treatment.After arthritis is induced 2 and remake the pathological observation of joint tissue after 3 months.The result shows (Fig. 3), and the single and cell mixed vaccine processed group is invaded profit and all obviously reduced than not treatment group.Presentation of results vaccine, especially mixed vaccine are lasting and significant to arthritic curative effect.
Embodiment 5: Mus MCP1 amino terminal hybridization peptide (N-MCP1-TT) and Mus MIP2 amino terminal hybridization peptide (N-MIP2-TT) mixed polypeptide vaccine are to the mouse arthritis therapeutic effect of stage of attack.
Three groups of mices, 16 every group, all as embodiment 3 usefulness fully not the formula adjuvant induce arthritis, but do not give any vaccine of mice.When arthritis has begun outbreak, second day that finds arthroncus begins to treat with vaccine immunity: one group of mixed vaccine of injecting N-MCP1-TT and N-MIP2-TT, in contrast, another group is only injected the vaccine of the special fragment of tetanus toxin (TT) preparation, also has one group of injection solvent (not treatment group).Method and dosage such as embodiment 3.Inject altogether twice.Fig. 4 result shows that mixed vaccine also has certain therapeutic effect to the arthritis that begins to show effect.On the contrary, continue with TT-vaccine processed group and the arthroncus of not treatment group.
Embodiment 6: the mixed polypeptide vaccine by Mus MCP1 amino terminal hybridization peptide (N-MCP1-DNA) and Mus MIP2 amino terminal hybridization peptide (N-MIP2-DNA) preparation suppresses mouse arthritis.
This test is adopted and with embodiment 3 identical methods and condition mice to be carried out arthritis and induce, and uses vaccine to treat.Unique difference is that the mixed polypeptide vaccine that adopts is to be prepared from by hybridization peptide N-MCP1-DNA and N-MIP2-DNA, rather than as the usefulness N-MCP1-TT of embodiment 3 and the mixed vaccine of N-MIP2-TT preparation.In other words, the difference of these two kinds of vaccines is that what to use among immunity identification fragment: the embodiment 3 of its Chemotactic Peptide is the special fragment (TT sees sequence 18) of tetanus toxin, and embodiment 6 usefulness is the dna fragmentation that contains CpG.Fig. 5 shows that this mixed vaccine is to similar with the mixed vaccine effect of N-MIP2-TT preparation by hybridization peptide N-MCP1-TT: this mixed vaccine can stop arthritic outbreak effectively.The result shows that also only the single vaccine with above-mentioned N-MCP1-DNA preparation also has a spot of inhibition effect.
Embodiment 7: produce specific antibody in Mus MCP1 amino terminal hybridization peptide (N-MCP1-TT) and the vaccine-induced body of Mus MIP2 amino terminal hybridization peptide (N-MIP2-TT) mixed polypeptide.
Four groups of mices are treated with the mixed polypeptide vaccine immunity through embodiment 3 described modes.Inducing arthritic the 20th day with the mice blood-letting, collecting and respectively organize serum.Mus MCP-1 and MIP-2 protein are connected on the integrated enzyme reaction lithographic plate.The serum of above-mentioned collection is done a series of dilution, be added to afterwards on the integrated enzyme reaction lithographic plate and combine test with MCP-1 or MIP-2 protein.The integrated enzyme reaction colour developing of the second antibody by the hydrogen peroxide enzyme labelling.What Fig. 6 expressed is the greatest dilution that can detect the antibodies reaction.The result shows, injection Mus MCP1 amino terminal hybridization peptide (N-MCP1-TT) can bring out mice specifically and produce the anti-proteinic antibody of self MCP-1, and same Mus MIP2 amino terminal hybridization peptide (N-MIP2-TT) can bring out and produce the anti-proteinic antibody of self MIP-2.It is special to the used chemotactic factor of vaccine bringing out, because the antibody that produces does not play association reaction to other protein, the antibody that brings out such as N-MCP1-TT does not combine with MIP-2, and mixed vaccine brings out two kinds of antibody, not only anti-MCP-1 but also anti-MIP-2.The result shows that the hybridization peptide vaccine is likely by destroying in the body self inflammatory cell chemotactic factor toleration, brings out producing corresponding specific antibody in the body.Vaccine brings out endocrine these antibody of body and can neutralize and remove in the body self excretory inflammatory cell chemokine protein matter, thereby has suppressed the invade profit of leukocyte to inflammation tissue, reaches the effect that reduces recurrence and treat autoimmune disease.
Embodiment 8: spontaneous dry syndrome and relevant chemotactic factor.
The spontaneous with advancing age generation dry syndrome of MRL mice, about 6 weeks morbidity, 12-14 week peaks.Show as the salivary gland inflammation, have a large amount of T lymphocytes and neutrophilic granulocyte invade profit (Fig. 7 a) causes the secreting gland necrosis, atrophy, thereby lose secretory function.Limited pcr analysis result shows that (method is with embodiment 3) expresses rising mainly is IP-10, ITAC, the gene of MCP-1 and MIP-2 (Fig. 7 b).
Embodiment 9: Mus IP-10 amino terminal hybridization peptide (N-IP10-TT) and Mus MIP2 amino terminal hybridization peptide (N-MIP2-TT) mixed polypeptide vaccine are to the treatment of dry syndrome.
4 weeks, big or small MRL mice was divided two groups in work, 25 every group.(dosage is 100 micrograms/0 to the mixed vaccine of subcutaneous injection solvent (matched group) or N-IP10-TT and N-MIP2-TT respectively.5 milliliters/only).Every partly measuring duplicate injection once (intramuscular), totally 4 times two weeks.In 4 weeks, 8,12,16 and 20 all every group kill 4 mices respectively, its salivary gland is done section statining.Fig. 8 shows that the mixed vaccine immunization therapy can alleviate the state of an illness of dry syndrome significantly, all is lower than matched group significantly up to the inflammation degree of the 20th all salivary gland of off-test.
The result who measures the treatment of (method is with embodiment 7) mixed polypeptide vaccine immunity by integrated enzyme reaction inducing mouse specifically produces the antibody of anti-self IP-10 and MIP-2, and does not induce anti-MCP-1 antibodies (Fig. 9).
Embodiment 10: Mus IP10 amino terminal hybridization peptide (N-IP10-TT) and Mus MIP2 amino terminal hybridization peptide (N-MIP2-TT) mixed polypeptide vaccine and Mus MCP1 amino terminal hybridization peptide (N-MCP1-TT) and Mus MIP2 amino terminal hybridization peptide (N-MIP2-TT) mixed polypeptide vaccine are to the treatment of systemic lupus erythematosus (sle).
The MRL mice another kind of autoimmune disease of spontaneous generation with advancing age is exactly a systemic lupus erythematosus (sle).Show as glomerulonephritis, albuminuria, kidney is depleted even dead.A large amount of mononuclear cells is found in inflammatory phase renal tissue dyeing, T lymphocyte and neutrophilic granulocyte invade profit.Approximately 8-9 week morbidity, mortality rate has reached 70 percent in the time of 6 months.In addition, lupus dermatitis is also very serious.12 weeks beginning mouse skin is obviously inflamed, even ulcer, and a large amount of leukocyte are invaded profit, mainly are T lymphocyte and neutrophilic granulocyte.
This experimental test the curative effect of mixed vaccine immunity to systemic lupus erythematosus (sle).4 weeks, big or small mice was divided three groups, 20 every group.Difference subcutaneous injection solvent (matched group) adds N-MIP2-TT with N-IP10-TT or N-MCP-TT adds two kinds of different mixed vaccines of N-MIP2-TT.Dosage is 100 micrograms/0.5 milliliters/only.Every partly measuring duplicate injection once (intramuscular), totally 4 times two weeks.The 8th, 12,16 and 20 all every group kill 5 mices respectively, its kidney and skin are done section statining.Figure 10 shows that up to the 20th week of off-test, compare not treatment group, two kinds of different mixed vaccine immunization therapies all reduce the invade profit of leukocyte to kidney and skin to a certain extent, and the state of an illness alleviates.
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.
Adnexa: the sequence/SEQ of patent thing (illustration is not limited thereto)
＜110〉Gong Xiaodi
＜120〉sequence of mixed polypeptide vaccine
<160>42
＜210〉1:N-Mcpl fragment
<211>19
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of MCP-1 is used for the chemotactic factor amino terminal fragment 1 that merges with immunity identification fragment
<400>1
Gln?Pro?Asp?Ala?Ile?Asn?Ala?Pro?Val?Thr?Cys?Cys?Tyr?Asn?Phe?Thr
15?10?15
Asn?Arg?Lys
19
＜210〉2:N-IP10 fragment
<211>17
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of IP10 is used for the chemotactic factor amino terminal fragment 2 that merges with immunity identification fragment
<400>2
Val?Pro?Leu?Ser?Arg?Thr?Val?Arg?Cys?Thr?Cys?Ile?Ser?Ile?Ser?Asn
15?10?15
Gln
17
＜210〉3:N-ITAC fragment
<211>17
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of ITAC is used for the chemotactic factor amino terminal fragment 3 that merges with immunity identification fragment
<400>3
Phe?Pro?Met?Phe?Lys?Arg?Gly?Arg?Cys?Leu?Cys?Ile?Gly?Pro?Gly?Val
15?10?15
Lys
17
＜210〉4:N-MIG fragment
<211>17
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of MIG is used for the chemotactic factor amino terminal fragment 4 that merges with immunity identification fragment
<400>4
Thr?Pro?Val?Val?Arg?Lys?Gly?Arg?Cys?Ser?Cys?Ile?Ser?Thr?Asn?Gln
15?10?15
Gly
17
＜210〉5:N-MCP3 fragment
<211>19
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of MCP-3 is used for the chemotactic factor amino terminal fragment 5 that merges with immunity identification fragment
<400>5
Gln?Pro?Val?Gly?Ile?Asn?Thr?Ser?Thr?Thr?Cys?Cys?Tyr?arg?Phe?Ile
15?10?15
Asn?Lys?Lys
19
＜210〉6:N-MCP4 fragment
<211>19
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of MCP-4 is used for the chemotactic factor amino terminal fragment 6 that merges with immunity identification fragment
<400>6
Gln?Pro?Asp?Ala?Leu?Asn?Val?Pro?Ser?Thr?Cys?Cys?Phe?Thr?Phe?Ser
15?10?15
Ser?Lys?Lys
19
＜210〉7:N-RANTES fragment
<211>10
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of RANTES is used for the chemotactic factor amino terminal fragment 7 that merges with immunity identification fragment
<400>7
Ser?Phe?Tyr?Ser?Ser?Asp?Thr?Thr?Pro?Cys
15?10
＜210〉8:N-MIP-1alpha fragment
<211>18
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of MIP-1alpha is used for the chemotactic factor amino terminal fragment 8 that merges with immunity identification fragment
<400>8
Ala?Ser?Leu?Ala?Ala?Asp?Thr?Pro?Thr?Ala?Cys?Cys?Phe?Ser?Tyr?Thr
15?10?15
Ser?Arg
18
＜210〉9:N-IL-8 fragment
<211>16
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of IL-8 is used for the chemotactic factor amino terminal fragment 9 that merges with immunity identification fragment
<400>9
Ser?Ala?lys?Glu?Leu?Arg?Cys?Gln?Cys?Ile?Lys?Thr?Tyr?Ser?Lys?Pro
15?10?16
＜210〉10:N-ENA78 fragment
<211>21
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of ENA78 is used for the chemotactic factor amino terminal fragment 10 that merges with immunity identification fragment
<400>10
Ala?Gly?Pro?Ala?Ala?Ala?Val?Leu?Arg?Glu?Leu?Arg?Cys?Val?Cys?Leu
15?10?15
Gln?Thr?Thr?Gln?Gly
20?21
＜210〉11:N-GRO alpha fragment
<211>17
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of GRO-alpha is used for the chemotactic factor amino terminal fragment 11 that merges with immunity identification fragment
<400>11
Ala?Ser?Val?Ala?Thr?Glu?Leu?Arg?Cys?Gln?Cys?Leu?Gln?Thr?Leu?Gln
15?10?15
Gly
17
＜210〉12:N-SLC fragment
<211>16
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of SLC is used for the chemotactic factor amino terminal fragment 12 that merges with immunity identification fragment
<400>12
Ser?Asp?Gly?Gly?Ala?Gln?Asp?Cys?Cys?Leu?Lys?Tyr?Ser?Gln?Arg?Lys
15?10?15
＜210〉13:N-ELC fragment
<211>17
<212>PRT
＜213〉people (human)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of ELC is used for the chemotactic factor amino terminal fragment 13 that merges with immunity identification fragment
<400>13
Gly?Thr?Asn?Asp?Ala?Glu?Asp?Cys?Cys?Leu?Ser?Val?Thr?Gln?Lys?Pro
15?10?15
IIe
17
＜210〉14:N-IP10 fragment
<211>17
<212>PRT
＜213〉Mus (mouse)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of mouse IP-10 is used for the amino end of chemotactic factor of merging with immunity identification fragment
End fragment 14
<400>14
Ile?Pro?Leu?Ala?Arg?Thr?Val?Arg?Cys?Asn?Cys?Ile?His?Ile?Asp?Asp
15?10?15
Gly
17
＜210〉15:N-MCP1 fragment (also claiming JE) fragment
<211>20
<212>PRT
＜213〉Mus (mouse)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of JE is used for the chemotactic factor amino terminal fragment 15 that merges with immunity identification fragment
<400>15
Gln?Pro?Asp?Ala?Val?Asn?Ala?Pro?Leu?Thr?Cys?Cys?Tyr?Ser?Phe?Thr
15?10?15
Ser?Lys?Met?Ile
20
＜210〉16:N-MIP2 fragment
<211>19
<212>PRT
＜213〉Mus (mouse)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of MIP-2 is used for the chemotactic factor amino terminal fragment 16 that merges with immunity identification fragment
<400>16
Ala?Val?Val?Ala?Ser?Phe?Leu?Arg?Cys?Gln?Cys?Leu?Lys?Thr?Leu?Pro
15?10?15
Arg?Val?Asp
19
＜210〉17:N-MCP5 fragment
<211>23
<212>PRT
＜213〉Mus (mouse)
<220>
<221>SITE
<222>
＜223〉the N-terminal fragment of mouse MCP-5 is used for the chemotactic factor amino terminal that merges with immunity identification fragment
Fragment 17
<400>17
Gln?Val?Leu?Ala?Gly?Pro?Asp?Ala?Val?Ser?Thr?Pro?Val?Thr?Cys?Cys
15?10?15
Tyr?Asn?Val?Val?Lys?Gln?Lys
20?23
＜210〉18: immunity identification fragment 1
<211>17
<212>PRT
＜213〉clostridium tetani (Clostridium tetani)
<220>
<221>SITE
<222>
＜223 〉, take from tetanus toxin, be used for merging immunity identification fragment 1 with chemotactic factor amino terminal fragment
<400>18
Gln?Tyr?Ile?Lys?Ala?Asn?Ser?Lys?Phe?Ile?Gly?Ile?Thr?Glu?Leu
15?10?15
Lys?Lys
17
＜210〉19: immunity identification fragment 2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc-feature
<222>
＜223〉fragment 2 is discerned in immunity, contains DNA (non-methylating) fragment-1 (CpG-DNA-1) of CpG
<400>19
tcc?atg?acg?atc?ctg?atg?ct??????20
＜210〉20: immunity identification fragment 3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc-feature
<222>
＜223〉fragment 3 is discerned in immunity, contains DNA (non-methylating) fragment-1 (CpG-DNA-1) of CpG
<400>20
tcc?atg?tcg?gtc?ctg?atg?ct??????20
＜210〉21: immunity identification fragment 4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc-feature
<222>
＜223〉fragment 4 is discerned in immunity, contains DNA (non-methylating) fragment-1 (CpG-DNA-1) of CpG
<400>21
tcc?atg?tcg?ctc?ctg?atg?ct????????20
＜210〉22: immunity identification fragment-5 (CT)
<211>240
<212>PRT
＜213〉cholera bacilli (Cholera)
<220>
<221>MUTAGEN
<222>
＜223〉immunity identification fragment-5 (CT), avirulent cholera bacilli toxin subunit (CTA1) fragment of modification is used for and chemotactic factor
The amino terminal fragment merges
<400>22
Asn?Asp?Asp?lys?Leu?Tyr?Arg?Ala?Asp?Ser?Lys?Pro?Pro?Asp?Glu?Ile
15?10?15
Lys?Gln?Ser?Gly?Gly?Leu?Met?Pro?Arg?Gly?Gln?Ser?Glu?Tyr?Phe?Asp
20?25?30
Arg?Gly?Thr?Gln?Met?Asn?Ile?Asn?Leu?Tyr?Asp?His?Ala?Arg?Gly?Thr
35?40?45
Gln?Thr?Gly?Phe?Val?Arg?His?Asp?Asp?Gly?Tyr?Val?Ser?Thr?Ser?Ile
50?55?60
Ser?Leu?Arg?Ser?Ala?His?Leu?Val?Gly?Gln?Thr?Ile?Leu?Ser?Gly?His
65?70?75?80
Ser?Thr?Tyr?Tyr?Ile?Tyr?Val?Ile?Ala?Thr?Ala?Pro?Asn?Met?Phe?Asn
85?90?95
Val?Asn?Asp?Val?Leu?Gly?Ala?Tyr?Ser?Pro?His?Pro?Asp?Glu?Gln?Glu
100?105?110
Val?Ser?Ala?Leu?Gly?Gly?Ile?Pro?Tyr?Ser?Gln?Ile?tyr?Gly?Trp?Tyr
115?120?125
Arg?Val?His?Phe?Gly?Val?Leu?Asp?Glu?Gln?Leu?His?Arg?Asn?Arg?Gly
130?135?140
Tyr?Arg?Asp?Arg?Tyr?Tyr?Ser?Asn?Leu?Asp?Ile?Ala?Pro?Ala?Ala?Asp
145?150?155?160
Gly?Tyr?Gly?Lle?Ala?Gly?Phe?Pro?Pro?Glu?His?Arg?Ala?Trp?Arg?Glu
165?170?175
Glu?Pro?Trp?Ile??His?His?Ala?Pro?Pro?Gly?Cys?Gly?Asn?Ala?Pro?Arg
180?185?190
Ser?Ser?Ile?Ser?Asn?Thr?Cys?Asp?Glu?Lys?Thr?Gln?Ser?Leu?Gly?Val
195?200?205
Lys?Phe?Leu?Asp?Glu?Tyr?Gln?Ser?Lys?Val?Lys?Arg?Gln?Ile?Phe?Ser
210?215?220
Gly?Lys?Gln?Ser?Asp?Ile?Asp?Thr?His?Asn?Arg?Ile?Lys?Asp?Glu?Leu
225?230?235?240
＜210〉23: immunity identification fragment 6, (LT)
<211>237
<212>PRT
＜213〉Escherichia coli (Escherichia coli)
<220>
<221>MUTAGEN
<222>
＜223〉immunity identification fragment 6 (LT) is taken from the thermo-responsive toxin of Escherichia coli, and modification avirulent is used for and chemotactic factor ammonia
The base terminal fragment merges
<400>23
Asn?Gly?Asp?Arg?Leu?Lys?Arg?Ala?Asp?Ser?Arg?Pro?Pro?Asp?Glu?Ile
15?10?15
Lys?Lys?Phe?Arg?Ser?Leu?Met?Pro?Arg?Gly?Gln?Asn?Glu?Tyr?Phe?Asp
20?25?30
Arg?Gly?Thr?Gln?Met?Asn?Ile?Asn?Leu?Tyr?Asp?His?Ala?Arg?Gly?Thr
35?40?45
Gln?Thr?Gly?Phe?Val?Arg?Tyr?Asp?Asp?Gly?Tyr?Val?Ser?Thr?Ser?Leu
50?55?60
Ser?Leu?Arg?Ser?Ala?His?Leu?Ala?Gly?Gln?Tyr?Ile?Leu?Ser?Gly?Tyr
65?70?75?80
Ser?Leu?Thr?Ile?Tyr?Ile?Val?Ile?Ala?Asn?Met?Phe?Asn?Val?Asn?Asp
85?90?95
Val?Ile?Ser?Val?Tyr?Ser?Pro?His?Pro?Tyr?Glu?Gln?Glu?Val?Ser?Ala
100?105?110
Leu?Gly?Gly?Ile?Pro?Tyr?Ser?Gln?Ile?Tyr?Gly?Trp?Tyr?Arg?Val?Asn
115?120?125
Phe?Gly?Val?Ile?Asp?Glu?Arg?Leu?His?Arg?Asn?Arg?Glu?Tyr?Arg?Asp
130?135?140
Arg?Tyr?Tyr?Arg?Asn?Leu?Asn?Ile?Ala?Pro?Ala?Glu?Asp?Gly?Tyr?Arg
145?150?155?160
Leu?Ala?Gly?Phe?Pro?Pro?Asp?His?Gln?Ala?Trp?Arg?Glu?Glu?Pro?Trp
165?170?175
Ile?His?His?Ala?Pro?Gln?Gly?Cys?Gly?Asp?Ser?Ser?Arg?Thr?Ile?Thr
180?185?190
Gly?Asp?Thr?Cys?Asn?Glu?Glu?Thr?Gln?Asn?Leu?Ser?Thr?Ile?Tyr?Leu
195?200?205
Arg?Glu?Tyr?Gln?Ser?Lys?Val?Lys?Arg?Gln?Ile?Phe?Ser?Asp?Lys?Gln
210?215?220
Ser?Glu?Val?Asp?Ile?Tyr?Asn?Arg?Ile?Arg?Asp?Glu?Leu
225?230?235????????????????????????????????????????????237
＜210〉24:N-Mcp1 fragment
<211>57
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of MCP-1 is used for the chemotactic factor amino terminal fragment that merges with immunity identification fragment
<400>24
cagccagatg?caatcaatgc?cccagtcacc?tgctgttata?acttcaccaa?taggaag?57
＜210〉25:N-IP10 fragment
<211>51
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of IP10 is used for the chemotactic factor amino terminal fragment 2 that merges with immunity identification fragment
<400>25
gtacctctct?ctagaaccgt?acgctgtacc?tgcatcagca?ttagtaatca?a?????????????51
＜210〉26:N-ITAC fragment
<211>51
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of ITAC is used for the chemotactic factor amino terminal fragment 3 that merges with immunity identification fragment
<400>26
ttccccatgt?tcaaaagagg?acgctgtctt?tgcataggcc?ctggggtaaa?a????????????51
＜210〉27:N-MIG fragment
<211>51
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of MIG is used for the chemotactic factor amino terminal fragment 4 that merges with immunity identification fragment
<400>27
accccagtag?tgagaaaggg?tcgctgttcc?tgcatcagca?ccaaccaagg?g?51
＜210〉28:N-Mcp3 fragment
<211>57
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of MCP-3 is used for the chemotactic factor amino terminal fragment 5 that merges with immunity identification fragment
<400>28
cagccagttg?ggattaatac?ttcaactacc?tgctgctaca?gatttatcaa?taagaaa?57
＜210〉29:N-MCP4 fragment
<211>57
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of MCP-4 is used for the chemotactic factor amino terminal fragment 6 that merges with immunity identification fragment
<400>29
cagccagatg?cactcaacgt?cccatctact?tgctgcttca?catttagcag?taagaag?57
＜210〉30:N-RANTES fragment
<211>30
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of RANTES is used for the chemotactic factor amino terminal fragment 7 that merges with immunity identification fragment
<400>30
tccccatatt?cctcggacac?cacaccctgc??30
＜210〉31:N-MIP-1alpha fragment
<211>54
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of MIP-1alpha is used for the chemotactic factor amino terminal fragment 8 that merges with immunity identification fragment
<400>31
gcatcacttg?ctgctgacac?gccgaccgcc?tgctgcttca?gctacacctc?ccgg?????????54
＜210〉32:N-IL-8 fragment
<211>48
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of IL-8 is used for the chemotactic factor amino terminal fragment 9 that merges with immunity identification fragment
<400>32
agtgctaaag?aacttagatg?tcagtgcata?aagacatact?ccaaacct????48
＜210〉33:N-ENA78 fragment
<211>63
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of ENA78 is used for the chemotactic factor amino terminal fragment 10 that merges with immunity identification fragment
<400>33
gctggtcctg?ccgctgctgt?gttgagagag?ctgcgttgcg?tttgtttaca?gaccacgcaa?60
gga???????????????????????????????????????????????????????????????63
＜210〉34:N-GRO-alpha fragment
<211>51
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of GRO-alpha is used for the chemotactic factor amino terminal fragment 11 that merges with immunity identification fragment
<400>34
gcgtccgtgg?ccactgaact?gcgctgccag?tgcttgcaga?ccctgcaggg?a????51
＜210〉35:N-SLC fragment
<211>48
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of SLC is used for the chemotactic factor amino terminal fragment 12 that merges with immunity identification fragment
<400>35
agtgatggag?gggctcagga?ctgttgcctc?aagtacagcc?aaaggaag?????????????????48
＜210〉36:N-ELC fragment
<211>51
<212>CDS
＜213〉people (human)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of ELC is used for the chemotactic factor amino terminal fragment 13 that merges with immunity identification fragment
<400>36
ggcaccaatg?atgctgaaga?ctgctgcctg?tctgtgaccc?agaaacccat?c???51
＜210〉37:N-MCP5 fragment
<211>69
<212>CDS
＜213〉Mus (mouse)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of mouse MCP-5 is used for the chemotactic factor amino terminal that merges with immunity identification fragment
Fragment 17
<400>37
caggtattgg?ctggaccaga?tgcggtgagc?accccagtca?cgtgctgtta?taatgttgtt??60
aagcagaag??????????????????????????????????????????????????????????69
＜210〉38:N-MCP1 fragment (also claiming JE) fragment
<211>60
<212>CDS
＜213〉Mus (mouse)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of JE is used for the chemotactic factor amino terminal fragment 15 that merges with immunity identification fragment
<400>38
cagccagatg?caatcaatgc?cccagtcacc?tgctgttata?acttcaccaa?taggaagatc???60
＜210〉39:N-IP10 fragment
<211>51
<212>CDS
＜213〉Mus (mouse)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of mouse IP-10 is used for the amino end of chemotactic factor of merging with immunity identification fragment
End fragment 14
<400>39
atccctctcg?caaggacggt?ccgctgcaac?tgcatccata?tcgatgacgg?g??51
＜210〉40:N-MIP2 fragment
<211>57
<212>CDS
＜213〉Mus (mouse)
<220>
<221>misc-feature
<222>
＜223〉the N-terminal fragment of MIP-2 is used for the chemotactic factor amino terminal fragment 16 that merges with immunity identification fragment
<400>40
gctgttgtgg?ccagtgaact?gcgctgtcaa?tgcctgaaga?ccctgccaag?ggttgac??57
＜210〉41: immunity identification fragment-5
<211>722
<212>DNA
＜213〉cholera bacilli (Cholera)
<220>
<221>mutation
<222>
＜223〉immunity identification fragment-5 (CT), avirulent cholera bacilli toxin subunit (CTA1) fragment of modification is used for and chemotactic factor
The amino terminal fragment merges
<400>41
Aatgatgata?agttatatcg?ggcagattct?agacctcctg?atgaaataaa?gcagtcaggt????60
Ggtcttatgc?caagaggaca?gagtgagtac?tttgaccgag?gtactcaaat?gaatatcaac????120
Ctttatgatc?atgcaagagg?aactcagacg?ggatttgtta?ggcacgatga?tggatatgtt????180
tccacctcaa?ttagtttgag?agtgcccact?tagtgggtca?aactatattg?tctggtcatt????240
ctacttatta?tatatatgtt?atagccactg?cacccaacat?gtttaacgtt?aatgatgtat????300
taggggcata?cagtcctcat?ccagatgaac?aagaagtttc?tgctttaggt?gggattccat????360
actcccaaat?atatggatgg?tatcgagttc?attttggggt?gcttgatgaa?caattacatc????420
gtaatagggg?ctacagagat?agatattaca?gtaacttaga?tattgctcca?gcagcagatg????480
gttatggatt?ggcaggtttc?cctccggagc?aragagcttg?gagggaagag?ccgtggattc????540
atcatgcacc?gccgggttgt?gggaatgctc?caagatcatc?gatcagtaat?acttgcgatg????600
aaaaaaccca?aagtctaggt?gtaaaattcc?ttgacgaata?ccaatctaaa?gttaaaagac????660
aaatattttc?aggctatcaa?tctgatattg?atacacataa?tagaattaag?gatgaattat????720
ga???????????????????????????????????????????????????????????????????722
＜210〉42: immunity identification fragment 6, the special fragment of the thermo-responsive toxin of avirulent Escherichia coli (LT) of modification
<211>710
<212>DNA
＜213〉Escherichia coli (Escherichia coli)
<220>
<221>mutation
<222>
＜223〉immunity identification fragment 6 (LT) is taken from the thermo-responsive toxin of Escherichia coli, and modification avirulent is used for and chemotactic factor amino
Terminal fragment merges
<400>42
atggcgacag?attataccgt?gctgactcta?gacccccaga?tgaaataaaa?cgtttccgga????60
gtcttatgcc?cagaggtaat?gagtacttcg?atagaggaac?tcaaatgaat?attaatcttt????120
atgatcacgc?gagaggaaca?caaaccggct?ttgtcagata?tgatgacgga?tatgtttcca????180
cttctcttag?tttgagaagt?gctcacttag?caggacagta?tatattatca?ggatattcac????240
ttactatata?tatcgttata?gcaaatatgt?ttaatgttaa?tgatgtaatt?agcgtataca????300
gccctcaccc?atatgaacag?gaggtttctg?cgttaggtgg?aataccatat?tctcagatat????360
atggatggta?tcgtgttaat?tttggtgtga?ttgatgaacg?attacatcgt?aacagggaat????420
atagagaccg?gtattacaga?aatctgaata?tagctccggc?agaggatggt?tacagattag????480
caggtttccc?accggatcac?caagcttgga?gagaagaacc?ctggattcat?catgcaccac????540
aaggttgtgg?agattcatca?agaacaatca?caggtgatac?ttgtaatgag?gagacccaga????600
atctgagcac?aatatatctc?agggaatatc?aatcaaaagt?taagaggcag?atattttcag????660
actatcagtc?agaggttgac?atatataaca?gaattcggga?tgaattatga???????????????710
Table 1 chemotactic factor determination of activity result
| Chemotactic factor or the segmental title of chemotactic factor amino terminal | With combining of chemokine receptors | The activity of chemotactic factor |
| MCP-1 | ????+ | ????+ |
| MCP-1 amino terminal fragment | ????- | ????- |
| IL-8 | ????+ | ????+ |
| IL-8 amino terminal fragment | ????- | ????- |
| GROα | ????+ | ????+ |
| GRO α amino terminal fragment | ????- | ????- |
| MIP-1α | ????+ | ????+ |
| MIP-1 α amino terminal fragment | ????- | ????- |
| RANTES | ????+ | ????+ |
| RANTES amino terminal fragment | ????- | ????- |
| BCA-1 | ????+ | ????+ |
| BCA-1 amino terminal fragment | ????- | ????- |
| IP10 | ????+ | ????+ |
| IP10 amino terminal fragment | ????- | ????- |
* The above results only is part embodiment of the present invention, and other chemotactic factor amino terminal fragment of the present invention is not reported one by one at this.
*: assay method: combine test with chemokine receptors, and chemotactic factor activity test (that is inducing cell nigration) institute employing method is all as J.Gong, et al, Journal of Biological Chemistry, 271:10521-10527,1996. is described.