CN1351661A - Compounds - Google Patents

Abstract

CASB619 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CASB619 polypeptides and polynucleotides in diagnostics, and vaccines for prophylactic and therapeutic treatment of cancers, particularly ovarian and colon cancers, autoimmune diseases, and related conditions.

Description

New compound

Invention field

The present invention relates to polynucleotide, refer to " CASB619 polynucleotide, their encoded polypeptides (being called " CASB619 polypeptide " here), reorganization material and their preparation method here.In yet another aspect, the present invention relates to use this peptide species and polynucleotide to comprise the particularly method of colorectal carcinoma and autoimmune disease and other associated conditions of treatment cancer.Again on the one hand, the present invention relates to use material provided by the invention to identify the method for agonist and antagonist/inhibitor, and with the method for the unbalance relevant illness of compounds identified treatment and CASB619 polypeptide.Again on the one hand, the present invention relates to detect the diagnostic test of the illness relevant with CASB619 polypeptide active or horizontal abnormality.

It is believed that, polypeptide of the present invention and polynucleotide are important pathogenic agent of antineoplastic specificity prevention or treatment immunity, because, with respect to normal cell, they are specific expressed or highly spend expression in tumour, therefore, can cause the destruction of tumour cell by antigen specific immune mechanism by target-seeking.They also can be used for the appearance of diagnosing tumour cell.In addition, their unconventionality expressions in some cases can cause autoimmunely induce, abnormal immune replys, this can correct by using identical polypeptide or polynucleotide carry out immunization.In this respect, most important biological activity is the antigen and the immunogen activity of polypeptide of the present invention.Polypeptide of the present invention also can have at least a other biological activity of CASB619 polypeptide, the target that this makes it can be used as the treatment that is different from immunne response or prevents to intervene.

Relate to the CASB619 polypeptide in one aspect of the present invention.This peptide species comprises the isolated polypeptide that contains following aminoacid sequence: have 70% identity at least with SEQ ID NO:2 full length sequence, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably aminoacid sequence of the identity of 97-99% at least.This peptide species comprises the amino acid whose polypeptide that contains SEQ ID NO:2.

Polypeptide of the present invention comprises that also its aminoacid sequence and SEQ ID NO:2 full length sequence have 70% identity at least, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably isolated polypeptide of the identity of 97-99% at least.This peptide species comprises the polypeptide of SEQ ID NO:2.

Polypeptide of the present invention also comprises the isolated polypeptide by the polynucleotide encoding that comprises the sequence that SEQ ID NO:1 is contained.

The present invention also provides a kind of immunogenic fragments of CASB619 polypeptide, i.e. the CASB619 polypeptide successive part, and it has identical or substantially the same immunogenicity activity with the polypeptide of the aminoacid sequence that contains SEQ ID NO:2.That is to say that this fragment (in case of necessity can with a kind of carrier coupling) can produce the immunne response that can discern the CASB619 polypeptide.This immunogenic fragments can comprise such as lacking the terminal leader sequence of N-, striding the CASB619 polypeptide of diaphragm area or the terminal anchor region of C-.One preferred aspect, consist essentially of all ectodomains of a peptide species according to the immunogenic fragments of CASB619 of the present invention, wherein this polypeptide and SEQ ID NO:2 have 70% identity at least on the whole length of SEQ ID NO:2, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably identity of 97-99% at least.

The peptide fragment that comprises the CASB619 epi-position generally comprises at least 7 of SEQ ID NO:2, preferred 9 or 10 continuous amino acids.Preferred epi-position is shown in SEQ ID NO:5 to SEQID NO:68.

The peptide that comprises these epi-positions is a preferred aspect of the present invention.Have the mimic epitopes (mimotope) of identical characteristics with these epi-positions and contain produce with the CASB619 molecule in the immunogen of these mimic epitopess of immunne response of epi-position cross reaction also be part of the present invention.

Therefore, the present invention includes the isolated peptides that contains these epi-positions itself and its mimic epitopes.Thereby mimic epitopes is meant and the enough similar entity that can be identified the antibody recognition of natural molecule of natural CASB619 epi-position; (Gheysen, H.M. etc., 1986, " as antigenic synthetic peptide ", Wiley, Chichester, Ciba fund symposial 119, p130-149; Gheysen, H.M., 1986, Molecular Immunology, 23,7,709-715), or with the suitable carrier coupling time, can produce entity with the antibody of natural molecule cross reaction.

The peptide mimic epitopes of above-mentioned epi-position can be used for special purpose by adding, lacking or replace selected amino acid.Therefore, peptide of the present invention can be modified being easy to and is connected with a kind of protein carrier.For example, for some Chemical bond method, need to comprise a terminal cysteine in the epi-position.In addition, combine with a kind of protein carrier, need comprise one, make the unconjugated free-end maintenance of peptide and combining of carrier proteins surface away from the hydrophobic end of peptide in conjunction with end for peptide.The conformational freedom that this has reduced peptide, the closely similar conformation of conformation that peptide is more likely presented in whole natural molecule structure, have with peptide.For example, peptide can be modified to and have a N-terminal halfcystine and the amidation tail that C-terminal is hydrophobic.In addition, can carry out the interpolation or the replacement of one or more amino acid whose D steric isomers, prepare a kind of useful derivative, be used for for example strengthening the stability of peptide.One skilled in the art will appreciate that these modified peptides or mimic epitopes can be the mimic epitopess of all or part of non-peptide, wherein the residue of Zu Chenging is not limited to 20 kinds of natural amino acids.In addition, can use technology known in the art to make it cyclisation, thereby the conformation of restriction peptide make it to be similar to the shape of peptide sequence in whole molecule the time.The preferred method of cyclisation peptide comprises that a pair of cysteine residues of adding is to form disulphide bridges.

And, one skilled in the art will recognize that mimic epitopes of the present invention or immunogen can be greater than above-mentioned epi-positions, thereby can comprise sequence disclosed herein.Therefore, mimic epitopes of the present invention can form by extending at one or two terminal N that adds other natural residue of certain number and/or C-terminal.The peptide mimic epitopes also can be the reverse sequence of native sequences, and its sequence direction is opposite; Perhaps sequence can be all or is formed (counter-rotating sequence, inverso sequence) by D-steric isomer amino acid to small part.And peptide sequence also can be the sequence of oppositely reversing, and wherein the sequence direction is opposite, and amino acid is the D-stereoisomer form.The advantage of these reverse or reverse-counter-rotating peptides is that it is nonself, therefore can overcome the self tolerance problem in the immunity system.

In addition, the peptide mimic epitopes can be by using and can identify with polypeptide bonded antibody of the present invention such as phage display technology (EP 0 552 267 B1).This technology produces the peptide sequence of the structure of a large amount of simulation native peptides, and these peptide sequences therefore can be in conjunction with the antibody of anti-native peptides, but not necessarily have the obvious sequence homology with natural polypeptides.The significant advantage of this method is to identify the peptide with strong immunogen characteristic, maybe can overcome the potential autoantigen tolerance problem that may occur when using native sequence polypeptide.In addition, this technology can be identified the recognition feature of each native peptides according to the total chemical characteristic in the mimic epitopes sequence of identification.

The covalent coupling of peptide and immunogenic carrier can be undertaken by mode well-known in the art.Therefore,, can utilize carbodiimide, glutaraldehyde or N-[γ-maleoyl butyryl oxygen for example for direct covalent coupling] succinimide ester, utilize commercial allos bifunctional linker commonly used, as CDAP and SPDP (according to manufacturer's explanation).After the linked reaction, immunogen can be removed and purifying easily by dialysis method, gel filtration method and fractional separation method etc.

It is well known to those skilled in the art being used for the immunogenic carrier of the present invention.It is auxiliary that the function of carrier provides cytokine, so that help to induce the immunne response of anti-peptide.The limiting examples that can be used for carrier of the present invention comprises: the purified protein derivative (PPD) of keyhole limpet hemocyanin (KLH), serum albumin such as bovine serum albumin (BSA), deactivation bacteriotoxin such as tetanus or diphtheria toxin (TT and DT) or its recombinant fragment (as 1 district of TT fragment C or the transit zone of DT) or tuberculin.Perhaps, mimic epitopes or epi-position can be directly and liposome vectors stop and close, it can also comprise can provide the T cell auxiliary immunogen.Preferred mimic epitopes is 1: 1 to 20: 1 to the ratio of carrier, preferably each carrier loaded 3-15 peptide.

In one embodiment of the invention, preferred carrier is the protein D (EP 0 594 610 B1) of hemophilus influenzae.Protein D is that the IgD of hemophilus influenzae is conjugated protein, applies for a patent (WO 91/18926,grant number EP 0 594 610 B1) by Forsgren.In some cases, for example in the former expression system of recombinant immune, may need to use the fragment of protein D, for example protein D 1/3^Rd(100 to 110 amino acid of N-terminal (GB9717953.5) that comprise protein D).

The another kind of preferred method of presenting peptide of the present invention is in recombinant fusion molecule.For example EP 0,421 635 B has introduced and has used chimeric hepadnavirus core antigen particles to present allogenic polypeptide with virus-like particle.Like this, immunogen of the present invention can be included in the peptide of presenting in the chimeric particle of being made up of hepatitis B virus core antigen.In addition, recombination fusion protein can comprise mimic epitopes of the present invention and carrier proteins, as influenza virus NS1.For any recombinant expression protein of forming the present invention's part, these immunogenic nucleic acid of encoding can also can be an aspect of of the present present invention.

Peptide used among the present invention can be synthetic easily by solid phase method well-known in the art.Suitable synthesizing can utilize T-boc or F-boc method to carry out.The cyclisation peptide can utilize well-known F-moc method and polyamide resin to synthesize by solid phase method in full-automatic instrument.Perhaps, one skilled in the art will recognize that and manually carry out the essential lab procedure of this method.The technology of solid phase synthesis and method can be referring to " solid-phase peptide be synthetic: practical approach ", E.Atherton and R.C.Sheppard, and Oxford University Press IRL publishes (1989).Perhaps, peptide can pass through recombinant methods, is included in the nucleic acid molecule of expressing the coding simulation epi-position in bacterium or the mammal cell line, then the mimic epitopes of purifying expression.Recombinant expressed peptide and proteic technology are known technologies in this area, can be referring to Maniatis, T., Fritsch, E.F. with " molecular cloning lab guides such as Sambrook, second edition " Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

Polypeptide of the present invention or immunogenic fragments can be " maturation " albumen forms, perhaps can be a kind of parts than large protein such as precursor or fusion rotein.Usually preferably include and contain secretion or leader sequence, presequence, the sequence that can assist purifying such as polyhistidine residue, or can in the reorganization preparation process, play the additional sequences of stabilization.And, also consider to add exogenous polypeptid or lipid tail or polynucleotide sequence to increase the immunogenicity ability of final molecule.

In one aspect, the present invention relates to the soluble fusion protein of genetic engineering preparation, this fusion rotein contains the various parts of the constant region of the heavy chain of a peptide species of the present invention or its fragment and each subclass immunoglobulin (Ig) (IgG, IgM, IgA, IgE) or light chain.Preferably, immunoglobulin (Ig) is the particularly constant region part of IgG1 of human IgG, occurs in hinge area and merge.In a special embodiment, the Fc part can be removed by introducing a cutting sequence simply, and this cutting sequence can be cut with blood clotting factor Xa.And, the present invention relates to prepare the method for these fusion roteins, and relate to their application in drug screening, diagnosis and treatment by genetic engineering.A further aspect of the present invention also relates to the polynucleotide of this fusion rotein of encoding.The example of fusion protein technology can be found in international patent application Nos.WO94/29458 and WO94/22914.

Protein can engage by chemical process, or with the formal representation of recombination fusion protein, this fusion rotein form can prepare in a kind of expression system with the high level of more non-fusion rotein.Merge part and can help to provide T to assist epi-position (immunity fusion part), preferably can be by the auxiliary epi-position of the T that the people discerns; The auxiliary epi-position (expression enhanser) of T that perhaps can help albumen to express with high yield than original recombinant protein.Merging part preferably is that a kind of immunity merges part, is again to express the enhanser part.

Merging part comprises from the protein D of hemophilus influenzae with from the non-structural protein NS 1 (hemagglutinin) of influenza virus.Another kind of fusion part is the albumen that is called as LytA.The preferred C-terminal part of using this molecule.LytA is from streptococcus pneumoniae, its synthetic a kind of N-acetyl-L-ala amide enzyme---LytA Ntn hydrolase (by lytA genes encoding { " gene " 43 (1986) 265-272 pages or leaves }), the latter is a kind of autolysin, its some key in the peptidoglycan skeleton of degrading specifically.The affinity with choline or some cholinomimetic such as DEAE is responsible in the proteic C-terminal of LytA zone.This specific character has been used to develop the intestinal bacteria C-LytA expression plasmid that can be used for expressing fusion protein.The purifying that contains the segmental hybrid protein of C-LytA at its aminoterminal has had introduction { " biotechnology " 10 volume (1992) 795-798 pages or leaves }.Can use in the LytA molecule repeating part of finding at C-terminal, from residue 178, residue 188-305 for example.

The present invention also comprises above-mentioned variant polypeptides, promptly replaces the polypeptide different with object of reference by conservative amino acid, and wherein a kind of residue is replaced by the residue that another kind has similar features.Typical this being substituted between the following amino acid: Ala, Val, Leu and Ile; Ser and Thr; Acidic residues Asp and Glu; Asn and Gln; Alkaline residue Lys and Arg; Or aromatic moieties Phe and Tyr.Particularly preferably be wherein several, 5-10,1-5,1-3,1-2 or 1 variant that amino acid is replaced, lacked or increase with any array mode.

Polypeptide of the present invention can be prepared in any suitable manner.This peptide species comprises the polypeptide of the polypeptide of isolating natural generation, the polypeptide of reorganization preparation, synthetic preparation or with the polypeptide of the combined preparation of these methods.The method for preparing this peptide species is known in this area.

On the other hand, the present invention relates to the CASB619 polynucleotide.These polynucleotide comprise the separation polynucleotide of the nucleotide sequence that contains the following polypeptide of encoding: these polypeptide and SEQ IDNO:2 full length amino acid sequence have at least 70% identity, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity.Polypeptide with identity of at least 97% is highly preferred, and the polypeptide of 98-99% identity is more highly preferred and have at least, and the polypeptide with at least 99% identity is that topnotch is preferred.These polynucleotide comprise the polynucleotide of the nucleotide sequence that the SEQ ID NO:1 of the polypeptide that contains coding SEQ ID NO:2 is contained.

Polynucleotide of the present invention comprise that also whole coding regions of containing with the nucleotide sequence of coding SEQ ID NO:2 polypeptide have at least 70% identity, preferred at least 80% identity, more preferably at least 90% identity, more preferably the separation polynucleotide of the nucleotide sequence of at least 95% identity.Polynucleotide with identity of at least 97% are highly preferred, and the polynucleotide of 98-99% identity are more highly preferred and have at least, and the polynucleotide with at least 99% identity are that topnotch is preferred.

Polynucleotide of the present invention also comprise the separation polynucleotide that contain following nucleotide sequence: described nucleotide sequence and SEQ ID NO:1 full length sequence have at least 70% identity, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity.Polynucleotide with identity of at least 97% are highly preferred, and the polynucleotide of 98-99% identity are more highly preferred and have at least, and the polynucleotide with at least 99% identity are that topnotch is preferred.These polynucleotide comprise the polynucleotide that contain SEQ ID NO:1 polynucleotide and the polynucleotide of SEQ ID NO:1.These polynucleotide can insert in suitable plasmid or the recombinant microorganism carrier, be used for immunity (referring to for example Wolffet al., Science 247:1465-1468 (1990); Corr et al., J.Exp.Med.184:1555-1560 (1996); Doe et al., Proc.Natl.Acad.Sci.93:8578-8583 (1996)).The present invention also provides and above-mentioned polynucleotide complementary polynucleotide.

The present invention also provides the CASB619 polynucleotide passage, and it has the immunogen characteristic identical with the polynucleotide of SEQ ID NO:1 when giving the experimenter with it.

The present invention also provides the polynucleotide of the immunogenic fragments of the above CASB619 polypeptide that defines of coding.

The nucleotide sequence of SEQ ID NO:1 and human chromosome 1 clone RP4-641D22 figure p13.1-13.3 (going into Tibetan AL157901) have homology.The nucleotide sequence of SEQ ID NO:1 is the cDNA sequence, comprises the sequence (Nucleotide 1 to 3043) of the polypeptide of 1013 the amino acid whose SEQ ID NO:2 that encode.The nucleotide sequence of the polypeptide of coding SEQ ID NO:2 can be identical with the contained polypeptid coding sequence of SEQ ID NO:1, perhaps it can be the sequence that is different from the contained sequence of SEQ ID NO:1, because the redundancy (degeneracy) of genetic code, its also encode polypeptide of SEQ ID NO:2.The polypeptide of SEQ ID NO:2 is not relevant with any known protein.

Expection preferred polypeptide of the present invention and polynucleotide also have similar biological function/characteristic with its homeopeptide and polynucleotide.And preferred polypeptide of the present invention, immune fragment and polynucleotide have at least a activity of SEQ ID NO:1 or SEQ ID NO:2.

The present invention also relates to part or other imperfect polynucleotide and peptide sequence, they are at first identified before definite corresponding total length SEQ ID NO:1 and SEQ ID NO:2 sequence.

Therefore, one aspect of the present invention provides isolating polynucleotide, it:

(a) comprise that the full length sequence with SEQ ID NO:3 has 70% identity at least, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably nucleotide sequence of the identity of 97-99% at least;

(b) full length sequence that has with SEQ ID NO:3 has 70% identity at least, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably nucleotide sequence of the identity of 97-99% at least;

(c) polynucleotide of SEQ ID NO:3; Or

(d) coding has 70% identity at least with the full length amino acid sequence of SEQ ID NO:4, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably nucleotide sequence of the polypeptide of the identity of 97-99% at least; And the polynucleotide of SEQ ID NO:3.

The present invention also provides a peptide species, it:

(a) comprise that the full length sequence with SEQ ID NO:2 has 70% identity at least, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably aminoacid sequence of the identity of 97-99% at least;

(b) full length amino acid sequence that has with SEQ ID NO:2 has 70% identity at least, preferred at least 80% identity, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably aminoacid sequence of the identity of 97-99% at least;

(c) comprise the amino acid of SEQ ID NO:4; With

(d) be the polypeptide of SEQ ID NO:4;

And by the polypeptide of the polynucleotide encoding that comprises the contained sequence of SEQ ID NO:3.

The nucleotide sequence of SEQ ID NO:3 and the peptide sequence of its coding are from EST (expressed sequence tag) sequence.One skilled in the art will appreciate that unavoidably has some nucleotide sequence reading mistake (seeing Adama, M.D.et al., Nature 377 (supp) 3,1995) in est sequence.Therefore, the nucleotide sequence of SEQ ID NO:3 and by the peptide sequence of its coding having aspect the sequence accuracy same interior in restriction.In addition, the peptide sequence of SEQ ID NO:3 coding comprises the zone with homology or identical or close homology of structural similitude albumen and/or structure extremely similar (as conserved amino acid difference).

Polynucleotide of the present invention can the use standard clone and triage techniques (for example see " molecular cloning lab guides such as Sambrook by the human colon cancer cell mRNA cDNA library acquisition of deriving, second edition " Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York (1989).Polynucleotide of the present invention also can maybe can use the commercial technology of knowing synthetic available from natural origins such as genome dna libraries.

When polynucleotide of the present invention were used for recombinant production polypeptide of the present invention, polynucleotide can comprise the encoding sequence of mature polypeptide itself; Or be positioned at the mature polypeptide encoded sequence of same frame with other encoding sequence, the example of other encoding sequence for a kind of leading or secretion sequence of coding, preceding-, former-or preceding former-proteic sequence or other any peptide moiety.For example, a kind of flag sequence that can promote the fusion polypeptide purifying of can encoding.In the preferred embodiment of this respect of the present invention, flag sequence is a kind of 6 Histidine peptides, is provided by the pQE carrier that (Qiagen Inc.), sees " PNAS " 86:821-824 such as Gentz, the introduction in (1989); Or a kind of HA peptide tail.These polynucleotide can also contain non-coding 5 ' and 3 ' sequence, as transcribe, the sequence of non-translated sequence, montage and poly-adenosine signal, ribosome bind site and sequence stable mRNA.

The further embodiment of the present invention comprises the polynucleotide of coded polypeptide variant, described variant comprises the aminoacid sequence of SEQ ID NO:2, wherein has several for example 5 to 10,1 to 5,1 to 3,1 to 2 or 1 amino-acid residues to replace, lack or increase with any array mode.

The polynucleotide identical or enough identical with the contained nucleotide sequence of SEQ ID NO:1 can be as the hybridization probe of cDNA and genomic dna or the primer that reacts as nucleic acid amplification (PCR), separating the full-length cDNA and the genomic clone of code book invention polypeptide, and separation and SEQ ID NO:1 have high sequence similarity other gene (the symbiosis homologue (paralogs) that comprising encodes derives from the people and from inhuman species directly to the gene of homologue (orthologs) and symbiosis homologue) cDNA and genomic clone.Generally, these nucleotide sequences are identical with object of reference sequence 70%, and preferred 80% is identical, and more preferably 90% is identical, and most preferably 95% is identical.Probe or primer generally comprise at least 15 Nucleotide, and preferably at least 30 Nucleotide can have at least 50 Nucleotide.Particularly preferred probe has 30 to 50 Nucleotide.Particularly preferred primer has 20 to 25 Nucleotide.Particularly, derive from isogenic animal polypeptide of sequence or polynucleotide and can be used as immunogen, obtain people's gene cross reactivity immunne response.

The polynucleotide of code book invention polypeptide comprise that the homologue that derives from inhuman species can obtain by the method that may further comprise the steps: screen suitable library with sequence or its segmental label probe with SEQ ID NO:1 under tight hybridization conditions; Separate and contain described polynucleotide sequence full-length cDNA and genomic clone.This hybridization technique is well known to those skilled in the art.Preferred tight hybridization conditions comprises that 42 ℃ are incubated overnight in containing the solution of following composition: salmon sperm DNA is sheared in the sex change of 50% methane amide, 5x SSC (150mM NaCl, 15mM Trisodium Citrate), 50mM sodium phosphate (pH7.6), 5x Denhardt ' s solution, 10% dextran sulfate and 20 micrograms/ml, then washs filter membrane in 0.1x SSC in the time of about 65 ℃.Therefore, the present invention also comprises the polynucleotide that screen suitable library acquisition with the sequence with SEQ ID NO:1 or its segmental label probe under tight hybridization conditions.

One skilled in the art will recognize that under many circumstances, isolating cDNA sequence is incomplete, wherein lack the terminal polypeptid coding area of cDNA 5 '.

To one skilled in the art, there is several method to use and to know, can be used for obtaining full length DNA s or extend short DNAs, for example (be seen in as Frohman etc. based on the method for terminal rapid amplifying (RACE) method of cDNA, " PNAS " 85:8998-9002,1988).The nearest modification of this technology, for example MarathonTM technology (Clontech Laboratories Inc.) has obviously been simplified the searching to longer cDNAs.At Marathon^TMIn the technology, cDNAs extractive mRNA preparation from a kind of tissue of selection, and connect a kind of " joint " sequence at each end.Use gene specific then with the increase 5 ' end of DNA of " losing " of special oligonucleotide combination the carrying out nucleic acid amplification (PCR) of joint.Use " nido " primer to repeat the PCR reaction then, nested primer promptly design with the inside annealed primer of amplified production (be generally with a kind of joint Auele Specific Primer of 3 ' annealed of joint sequence and with a kind of gene-specific primer of 5 ' annealed of selected gene order).With dna sequencing the product of this reaction is analyzed then, produced a complete sequence, perhaps use the new sequence information of design 5 ' primer to carry out an independent total length PCR, make up the DNA of a total length by product directly is connected with existing DNA.

Recombinant polypeptide of the present invention can be by method well known in the art by the genetically engineered host cell preparation that comprises expression system.Therefore, on the other hand, the present invention relates to comprise the expression system of polynucleotide of the present invention, relate to the host cell of this expression system genetic modification with by recombinant technology and produce polypeptide of the present invention.Cell free translation system also can be used for producing these albumen, utilizes the RNA derived from DNA construct of the present invention.

For carrying out the reorganization preparation of polypeptide of the present invention, host cell can be by genetically engineered expression system or its part of integrating polynucleotide of the present invention.Polynucleotide are imported host cell can be finished by the method for multiple standards laboratory manual introduction, " molecular cloning lab guide such as Davis etc. " molecular biology basic skills " (1986) and Sambrook for example, second edition " Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYork (1989).Preferred this method comprises that transfection, transposition (transvection), microinjection, the transfection of cationic lipid mediation, electroporation, transduction, the cut of for example calcium phosphate transfection, the mediation of DEAE-dextran are inoculated, bombardment imports or infects.

Preferred albumen of the present invention and trans Trx (TIT) coexpression.Compare with the cis coexpression, trans Trx coexpression is preferred, does not have Trx in the antigen because it can make, and need not proteolytic enzyme.The Trx coexpression makes albumen of the present invention dissolve easily.The Trx coexpression also has remarkably influenced to protein purification rate, purifying protein solvability and quality.

The representative example of suitable host comprises bacterial cell such as suis, staphylococcus, intestinal bacteria, streptomycete, bacillus subtilis mycetocyte; Fungal cell such as yeast cell and aspergillus cell; The cell of insect cell such as fruit bat S2 and fall army worm Sf9; Zooblast such as CHO, COS, HeLa, C127,3T3, BHK, 293, CV-1 and Bowes melanoma cells; And vegetable cell.

Can use multiple expression system, for example karyomit(e), episome and viral flavor, for example by bacterial plasmid, bacteriophage, transposon, yeast episome, insertion element, yeast chromosomal element, virus as baculovirus, papovavirus such as SV40, vaccinia virus, adenovirus, fowlpox virus, Pseudorabies virus and retrovirus deutero-carrier or by their composition deutero-carrier, for example by plasmid and phage genetic elements deutero-carrier, as clay and phagemid.Expression system can contain the control area of regulating and causing expression.In general, anyly be suitable for keeping, breed or express polynucleotide produce polypeptide in the host system or carrier and all can use.Suitable nucleotide sequence can be inserted in the expression system by any technology with routine that know, for example the technology listed in (above) of Sambrook etc. " molecular cloning lab guide ".Suitable secretion signal can be incorporated in the target polypeptides, make the translation protein excretion in endoplasmic, in periplasmic space or the born of the same parents' external environment.These signals can be endogenic concerning polypeptide, perhaps also can be heterologys.

Expression system is a kind of live microorganism of reorganization also, for example a kind of virus or bacterium.Target gene can be inserted in the genome of recombinant virus alive or bacterium.The carrier that infects this work in inoculation and the body will cause antigenic expression in vivo and immune response inducing.The virus and the bacterium that are used for this purpose are such as poxvirus (for example vaccinia virus, fowlpox virus, canary avipoxvirus), Alphavirus (sindbis alphavirus, Semliki Forest virus, peste loca virus), adenovirus, adeno associated virus, picornavirus (poliovirus, rhinovirus), simplexvirus (varicella zoster virus etc.), listeria spp, Salmonellas, Shigellae, BCG.These are viral and bacterium can be toxic or the attenuation that ins all sorts of ways obtains a kind of living vaccine.This living vaccine also is a part of the present invention.

Polypeptide of the present invention can reclaim and purifying from the reconstitution cell culture by the technology of knowing, and these technology comprise ammonium sulfate or ethanol sedimentation, sour extracting, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography.Most preferably metal ion affinity chromatography (IMAC) is used for purifying.When polypeptide is synthetic in born of the same parents, in separation and/or the purge process during sex change, can use know be used for the folding again technology regeneration activity conformation of albumen.

Another importance of the present invention relates in Mammals the method for inducing, strengthening or regulate immunne response, comprise with polypeptide of the present invention or polynucleotide or its fragment seeded with mammalian, present in an amount at least sufficient to produce the antibody and/or the T cellullar immunologic response of prevention or treatment cancer and autoimmune disease and associated conditions.Another aspect of the present invention involves in the Mammals method of inducing, strengthening or regulate immunne response, the carrier or the cell that comprise the polynucleotide expression in vivo by instructing code book invention polypeptide give polypeptide of the present invention, thereby induce immune response produces the immunne response of preventing or treating the described disease of Mammals.

A further aspect of the present invention relates to a kind of immunity/vaccine preparation (composition), when it is imported mammalian hosts, induce, strengthen or regulate in the Mammals immunne response at polypeptide of the present invention, wherein composition comprises its immune fragment of polypeptide of the present invention or polynucleotide or this paper definition.Vaccine preparation can also comprise suitable carriers.Because polypeptide can be degraded under one's belt, preferably with its administration (as subcutaneous, muscle, intravenously or intradermal injection) outside enteron aisle.The preparation that is suitable for parenteral admin comprises water-based and non-aqueous aseptic parenteral solution, and it can contain antioxidant, buffer reagent, antiseptic-germicide and make preparation and the isoosmotic solute of recipient's blood; And the water-based and the non-aqueous sterile suspension that can comprise suspension agent or thickening material.Preparation can place in the container of single dose or multiple doses, Mi Feng peace bottle and bottle for example, and can be stored in cryodesiccated environment, only need add aseptic liquid vehicle before use.

The present invention relates to external evoked at polypeptide of the present invention or polynucleotide or its fragment or contain the immunne response of the molecule of polypeptide of the present invention or polynucleotide on the other hand, use the immune system cell, these activating immune cells are imported Mammals again with the treatment disease.The activation of immune system cell can be by when various immune modulatory molecules exist or do not exist, and external incubation complete polypeptide of the present invention or polynucleotide or the molecule that contains polypeptide of the present invention or polynucleotide are realized.The present invention relates on the other hand by giving antigen presenting cell immune Mammals, and this antigen presenting cell is modified by external load polypeptide of the present invention or its part or the molecule that contains polypeptide of the present invention, and with immunogenicity approach vivo medicine-feeding.Perhaps, antigen presenting cell can be with the carrier in-vitro transfection that contains polynucleotide of the present invention or fragment or contain polynucleotide of the present invention expressing corresponding polypeptide, and with immunogen mode vivo medicine-feeding.

Vaccine preparation of the present invention also can comprise adjuvant system, is used for strengthening the immunogenicity of preparation.The preferred adjuvant system preferentially causes the TH1 type and replys.

Immunne response can be divided into two typical kinds substantially, i.e. body fluid or cell-mediated immune responses (the general antibody and the cytological effect mechanism that shield used is respectively distinguished).These are replied kind and are called as the TH1 type and reply (cell-mediated replys) and TH2 type immunne response (humoral response).

The feature of typical TH1 type immunne response is cytotoxic T cell and the natural killer type cell response that produces the haplotype restriction of antigen-specific.In mouse, the feature that the TH1 type is replied normally produces the antibody of IgG2a hypotype, and in the people, their counterpart is an IgG1 type antibody.The feature of TH2 type immunne response is the immunoglobulin (Ig) isotype that produces wide spectrum, comprises IgG1, IgA and IgM in mouse.

Can imagine to obtain that the motivating force after this immunne response of two types is a cytokine.High-caliber TH1 cytokines is preferentially induced at the antigenic cell-mediated immune responses of give, and high-caliber TH2 cytokines is preferentially induced at antigenic humoral immunoresponse(HI).

The differentiation of TH1 and TH2 type immunne response is not absolute.In fact, body can be supported the immunne response of a kind of TH1 of being described to advantage or TH2 advantage one by one.But, usually easily according to Mosmann and Coffman the introduction in mouse CD4+ve T cell clone consider the (Mosmann of family of cytokine, T.R. and Coffman, R.L. (1989), TH1 and TH2 cell: the different secretion patterns of lymphokine cause different functional performances." immunology yearbook " 7 volumes, the 145-173 page or leaf).Usually, the TH1 type is replied relevant with the IL-2 cytokine with T lymphocyte generation INF-γ.Other usually directly can't help the generation of T cell with TH1 type immune response inducing related cytokine such as IL-12.On the contrary, the TH2 type is replied relevant with the secretion of IL-4, IL-5, IL-6 and IL-13.

Known particular vaccine adjuvant is particularly suitable for stimulating TH1 or TH2 cytokines to reply.The TH1 that vaccine inoculation or premunition are replied: TH2 equilibrated optimal parameter is usually included in external use antigen stimulates the back directly to measure TH1 or the TH2 cytokine that the T lymphocyte produces again, and/or measures the IgG1 of the antibody response of antigen-specific: the IgG2a ratio.

Therefore, TH1 type adjuvant is the adjuvant that the preferential immunoglobulin (Ig) that stimulates isolating T cell colony to produce high-caliber TH1 cytokines and promotion CD8+ cytotoxic T cell and the antigen-specific relevant with TH1 type isotype is replied generation when external use antigen stimulates again.

Can preferentially stimulate the adjuvant of TH1 cell response in international patent application No.WO94/00153 and WO95/17209, to introduce.

The single phosphinylidyne lipoid A of 3 De-O-acidylates (3D-MPL) are a kind of such adjuvants.This can be known by GB2220211 (Ribi).It is at the mixture that chemically is single phosphinylidyne lipoid A of 3 De-O acidylates and 4,5 or 6 acyl chains, and by Ribi Immunochem, Montana makes.A kind of preferred form of the single phosphinylidyne lipoid of 3 De-O-acidylates A is open inEuropean patent 0 689 454 (SmithKline Beecham Biologicals SA).

The 3D-MPL particle preferably is small enough to carry out filtration sterilization (European patent 0 689 454) by one 0.22 μ m millipore filtration.

3D-MPL is with every dose 10 μ g-100 μ g, and preferably the scope of 20-25 μ g exists, and antigen exists with the scope of every dose of 2-50 μ g usually.

Another preferred adjuvants comprises QS21, and it is a kind of nontoxic component of Hplc purifying of the stem from Quillaja SaponariaMolina.It is used for mixing with the single phosphinylidyne lipoid A of 3 De-O-acidylates (3D-MPL) alternatively, and uses with a kind of carrier alternatively.

The method for preparing QS21 is in U.S. Patent No. 5,057, and is open in 540.

Contain the former existing introduction of non-reacted adjuvant formulation (WO96/33739) of QS21.Contain QS21 and cholesteric this preparation and when preparing, shown it is that successful TH1 stimulates adjuvant with antigen.

The further adjuvant of preferential stimulation TH1 type cell response comprises the oligonucleotide of immunomodulatory, and for example non-methylated CpG sequence is as disclosing among the WO 96/02555.

The combination of the adjuvant that different TH1 pungency adjuvants are introduced as mentioned also is considered to provide a kind of adjuvant of preferential stimulation TH1 type cell response.For example, QS21 can with the 3D-MPL formulated in combination.The ratio of OS21: 3D-MPL is generally 1: 10 to 10: 1, is preferably 1: 5 to 5: 1, and is typically about 1: 1.Preferred best of breed scope is 2.5: 1 to 1: 1 3D-MPL: QS21.

According to the present invention, vaccine composition also preferably contains a kind of carrier.Carrier can be a kind of oil-in-water emulsion, or a kind of aluminium salt such as aluminum phosphate or aluminium hydroxide.

Oil-in-water emulsion preferably comprises a kind of metabolizable oil, for example squalene, alpha-tocopherol and Tween 80.One particularly preferred aspect, according to the present invention, antigen in the vaccine composition and QS21 and 3D-MPL make up in a kind of like this emulsion.In addition, this oil-in-water emulsion can contain span 85 and/or Yelkin TTS and/or tricaprylin.

To people's administration the time, the scope of QS21 and 3D-MPL is every dose 1 μ g-200 μ g in the vaccine, for example 10-100 μ g, preferably 10 μ g-50 μ g.Oil-in-water contains 2 to 10% squalene, 2 to 10% alpha-tocopherol and 0.3 to 3% Tween 80 usually.When providing with more stable emulsion form, the ratio of squalene: alpha-tocopherol: Tween 80 is preferred identical or be less than 1.The Span85 that also can comprise 1% level.In some cases, vaccine of the present invention preferably further contains a kind of stablizer.

Atoxic oil-in-water emulsion preferably contains a kind of atoxic oil such as squalane or squalene, a kind of emulsifying agent such as Tween 80 in a kind of water carrier.Water carrier can be a phosphate-buffered saline for example.

Introduced a kind of strong especially adjuvant formulation among the WO 95/17210, it contains QS21,3D-MPL and tocopherol in a kind of oil-in-water emulsion.

The present invention also provides a kind of polyvalent vaccine composition, and it contains vaccine preparation of the present invention and other antigen, particularly to treatment cancer, autoimmune disease and the useful antigen of associated conditions.A kind of like this multivalent vaccine composition can comprise a kind of foregoing TH1 induction type adjuvant.

The present invention also relates to polynucleotide and the polypeptide purposes as diagnostic reagent, polynucleotide are the primer form derived from polynucleotide of the present invention, and polypeptide is special antibody of polypeptide of the present invention or reagent form.

Can detect the optimal treatment that the evaluation of heredity that the oncogenesis approach changes extremely in early days or biochemical marker can help definite patient in blood or the tissue.Substitute tumor-marker such as polynucleotide and express the multi-form and state that can be used for diagnosing cancer.The expression level of identifying polynucleotide of the present invention can be used for distinguishing the stage of cancer and to the character classification of cancerous tissue.Whether distinguishing carcinoma stage is the progress of monitoring cancer, according to existing malignant tissue to determine in the examination of living tissue zone.Polynucleotide of the present invention are tested and appraised the invasive cancer sign as the existence of different zones in vivo, can help to improve the method in differentiation stage.The cancer classification shows the degree of approximation of tumour and its similar healthy tissues, is evaluated by cellular form and other differentiation sign.Polynucleotide of the present invention can be used for determining the tumour rank, because they can help to determine the differentiation state of tumour cell.

Diagnostic test is by comprising the diagnosis of determining from the method for unusual polypeptide that reduces or increase or mRNA level in experimenter's the sample, diagnosis is provided or determined method to the susceptibility of cancer, autoimmune disease and associated conditions.This diagnostic method is known to be expression of differentiation.The relatively expression of specific gene in diseased tissue and the healthy tissues.Polynucleotide genes involved, mRNA or the difference of albumen aspect molecular weight, amino acid or nucleotide sequence or relative abundance in two kinds of tissues show in the tissue of suspecting ill people gene or regulate and control the variation of its gene.

The expression that reduces or increase can be measured at rna level.At first from two kinds of tissues, separate PolyA RNA; use RNA trace or any other the direct or indirect RNA detection method that contains Poly A+mRNA then; by for example tissue slice in situ hybridization, reverse transcriptase PCR etc., can detect the mRNA of the genes encoding of the polynucleotide of the present invention that are equivalent to differential expression.The expression of comparing given RNA in the diseased tissue with healthy tissues increases or reduces, and shows that transcript and/or expressed proteins work in disease.Therefore, detecting the mRNA corresponding to SEQ ID NO:1 or 3 that more normally organizes high or low level shows have cancer in the patient.

The mRNA expression level can be determined from expressed sequence tag (EST) library of sample by producing in the sample.The relative appearance of EST can be used for estimating the relative appearance of gene transcripts in initial sample in the library.This EST analyzes experiment and can compare with the EST analysis of reference sample, thereby determines relative expression's level of herbicide-tolerant polynucleotide.

Other mRNA analyzes can use serial analysis of gene expression (SAGE) method (Velculescu et al., Science (1995) 270-484), differential method (for example US5,776,683) or depend on the Nucleotide specific hybridization analysis that interacts and carries out.

Perhaps, relatively can carry out at protein level.Albumen size in two kinds of tissues can use antibody relatively, detects polypeptide in the western blotting experiment from the protein extract of two kinds of tissues.Expression level and Subcellular Localization also can be used at the antibody mediated immunity of corresponding protein and measure.Those skilled in the art know other can be used for measuring experimental technique from albumen in a host's the sample such as polypeptide level of the present invention.Compare in the diseased tissue expression of polypeptides level raises or reduces and show that the expression of expression may be relevant with disease with healthy tissues.

In experiment of the present invention, diagnosis can be determined by the expression level of measuring the gene product of at least one sequence encoding shown in the SEQ ID NO:1 or 3.Morbid state and healthy tissues in mRNA or protein level more also can be used for follow the trail of disease process or disappear.

Multiple polynucleotide sequence in the sample can use the polynucleotide array analysis.These can be used to disclose the differential expression of gene and determine gene function.For example, SEQ ID NO:1 or 3 polynucleotide sequence array can be used for determining in normal and the cancerous issue whether any polynucleotide differential expression is arranged.In one embodiment, can make up the nucleotide sequence or its segmental oligonucleotide probe array that contain SEQ ID NO:1 or 3, so that effectively screen genetic mutation.Array technique is well-known, can widespread use, and can be with the various problems that solves in the molecular genetics, comprise that genetic expression, genetic linkage and hereditary mutability are (referring to for example M.Chee et al., Science, Vol 274, pp610-613 (1996)).

" diagnosis " comprises the susceptibility of definite experimenter to disease in the present invention, determines whether the experimenter is ill, and definite patient's prognosis.

The invention still further relates to the diagnostic kit that carries out diagnostic test, it comprises:

(a) polynucleotide of the present invention, preferred SEQ ID NO:1 or 3 nucleotide sequence or its fragment;

(b) with (a) complementary nucleotide sequence;

(c) polypeptide of the present invention, preferred SEQ ID NO:2 or 4 polypeptide or its fragment; Or

(d) antibody of polypeptide of the present invention, the antibody of preferred SEQ ID NO:2 or 4 polypeptide.

Nucleotide sequence of the present invention is also valuable to chromosomal localization.On this sequence-specific target human chromosome specific position and with its hybridization.Is the important the first step that these sequences and gene-correlation disease are connected according to the present invention to the mapping of karyomit(e) correlated series.Sequence by the accurate position on the karyomit(e) of mapping after, the physical location of sequence on karyomit(e) can connect with the genetic map data.These data can be referring to for example V.McKusick, MendelianInheritance in Man (by the online acquisition of Jhon Hopkins University Welch medical library).The gene of the identical chromosomal region of Australia of having been mapped then and the relation of disease can be passed through linkage analysis (the common heredity of the adjacent gene of physics) and identify.Also can determine cDNA or the adjacent difference of genome between the influenced and uninfluenced individuality.

Polypeptide of the present invention and its fragment or analogue or the cell of expressing this polypeptide also can be used as immunogen, and generation is to the specific antibody of polypeptide immune of the present invention." immunologic opsonin " be meant antibody to the avidity of polypeptide of the present invention obviously greater than its avidity to other related polypeptide of prior art.

The present invention provides polypeptide of the present invention or the antibody of its immune fragment immunologic opsonin that limits on the other hand herein.Preferred this antibody is monoclonal antibody.

At the antibody of polypeptide of the present invention also can by conventional route to animal preferably non-human animal's fragment, analogue or cell of giving polypeptide or having an epi-position obtain.In order to prepare monoclonal antibody, provide any technology of the antibody that produces by the continuous cell line culture all can use.Example comprises hybridoma technology (Kohler, G. and Milstein, C. " nature " 256:495-497 (1975)), three knurls (trioma) technology, human B cell hybridoma technology (" immunology today " 4:72 such as Kozbor (1983)) and EBV hybridoma technology (Cole etc. " monoclonal antibody and cancer therapy ", the 77-96 page or leaf, Alan R.Liss, Inc., 1985).

The technology (U.S. Patent No. 4,946,778) of preparation single-chain antibody can be revised the single-chain antibody for preparing at polypeptide of the present invention.Transgenic mouse or other biological body or animal comprise that other Mammalss also can be used to express humanized antibody.

Above-mentioned antibody can be used for separating or identify the clone of express polypeptide or pass through the affinitive layer purification polypeptide.Antibody of the present invention also can be used for preventing or treating cancer, particularly ovarian cancer and colorectal carcinoma, autoimmune disease and associated conditions.

Others of the present invention relate to the method for inducing or regulating immunne response in Mammals, comprise with peptide vaccination Mammals of the present invention, the antibody and/or the T cellullar immunologic response that present in an amount at least sufficient to produce protection or alleviate disease symptoms or process.Another aspect of the present invention relates to the method for inducing or regulating immunne response in Mammals; comprise that the carrier by instructing polynucleotide expression of the present invention and coded polypeptide in the body gives polypeptide of the present invention, produces antibody with induce immune response and protects this animal to avoid this disease.

Therefore, should recognize, the invention provides that a kind of treatment exists with the CASB619 polypeptide active, excessive or lowly relevant unusual illness such as cancer and the autoimmune disease method of ovarian cancer and colorectal carcinoma particularly.

The present invention also provides a kind of SCREENED COMPOUND to identify stimulates or suppresses the method for the compound of CASB619 polypeptide function.Generally, agonist or antagonist can be used for above-mentioned treatment of diseases and prevention purpose.Can be from multiple source authenticating compound, for example cell, acellular prepared product, chemical library and natural product compound.The agonist of Jian Dinging, antagonist or inhibitor according to circumstances can be the natural of polypeptide or modify substrate, part, acceptor, enzyme etc. like this; Perhaps can be its structure or functional analogue thing (seeing Coligan et al " Immunization Update method " 1 (2) the 5th chapter (1991)).Screening method is well known to those skilled in the art.Other screening method can be referring to for example D.Bennett et al., J Mol Recognition, 8:52-58 (1995); And K.Johanson et al., J Biol Chem, 270916): 9459-9471 (1995) and reference wherein.

Therefore, the invention provides a kind of screening method, comprise the following method that is selected to identify the compound that stimulates or suppress polypeptide function of the present invention:

(a), measure the combination of candidate compound to polypeptide (or the cell of load polypeptide or film) or its fusion rotein by direct or indirect and candidate compound bonded mark;

(b) in the presence of mark competition thing, measure the combination of candidate compound to polypeptide (or the cell of load polypeptide or film) or its fusion rotein;

(c) use is to the cell or the suitable detection system of cytolemma of load polypeptide, and whether the test candidate compound causes the polypeptide activation or suppress the signal of generation;

(d) solution of candidate compound with the polypeptide that contains claim 1 is mixed, form mixture, measure the activity of polypeptide in the mixture, relatively the activity of mixture and standard substance; Or

(e) use as the ELISA experiment waits the influence of the generation of the mRNA that measures this polypeptide of coding in the candidate compound pair cell and this polypeptide.

Polypeptide of the present invention can be used for by the combination of receptors bind technical evaluation film or the soluble acceptor of this area standard.The screening method of knowing also can be used for identifying the agonist and the antagonist of competition and polypeptide receptor bonded of the present invention polypeptide of the present invention.

Therefore, on the other hand, the present invention relates to identify the agonist, antagonist, part, acceptor, substrate, enzyme etc. of polypeptide of the present invention or reduce or improve the screening reagent box of the compound that these polypeptide produce that it comprises:

(a) polypeptide of the present invention;

(b) reconstitution cell of expression polypeptide of the present invention;

(c) cytolemma of expression polypeptide of the present invention; Or

(d) antibody of polypeptide of the present invention;

This polypeptide is the polypeptide of SEQ ID NO:2 preferably.

Those skilled in the art recognize that easily polypeptide of the present invention also can be used for the method based on structure design polypeptide agonist, antagonist or inhibitor, and is specific as follows:

(a) three-dimensional structure of at first definite polypeptide;

(b) infer the similar reaction of agonist, antagonist or inhibitor or the three-dimensional structure of binding site;

(c) synthetic expection and combining of inferring or reaction site in conjunction with or the candidate compound that reacts;

(d) whether the test candidate compound is agonist, antagonist or inhibitor really.

Gene therapy also can be used for influencing the endogenous generation by the CASB619 polypeptide of experimenter's relevant cell.The general introduction of gene therapy is referring to " human molecular genetics " the 20th chapter gene therapy and other treatment approach based on molecular genetics (and reference wherein), TStrachan and A P Read, BIOS Scientific Publishers Ltd (1996).

The general description of vaccine preparation is seen " medicine biotechnology " 61 volumes, vaccine design-subunit and adjuvant approach, and Powell and Newman compiles, Plenum Press, 1995.Vaccine new trend and development, volumes such as Voller, University Park Press, Baltimore, Maryland, USA.Being encapsulated in can be referring to for example Fullerton United States Patent (USP) 4,235,877 in the liposome.Albumen stops and is incorporated into macromole referring to for example United States Patent (USP)s 4,474,757 such as Likhite United States Patent (USP) 4,372,945 and Armor.

Protein content is replied for the protection of induction of immunity in typical vaccine recipient in selected every vaccinating agent does not have significant side effects.This amount depends on the concrete immunogen of use.Generally, expect that every dose contains 1-1000 μ g albumen, preferred 2-100 μ g, most preferably 4-40 μ g.The optimal dose of specific vaccine can be observed among antibody titers and the experimenter other research on standard of replying and determines by relating to.After the initial inoculation, the experimenter generally accepts booster immunization in about 4 weeks.

" isolating " expression " by artificial " changes its native state, if promptly " isolating " composition or material exist at occurring in nature, then it changes or take out or taken out and change from its primal environment.For example, when using this term in this article, polynucleotide or the polypeptide that exists in moving object is not " isolating ", but with native state under the material that coexists the as much Nucleotide or the polypeptide that separate be exactly " isolating ".

" polynucleotide " refer generally to any poly-ribonucleotide or poly-deoxyribonucleotide, and it can be the RNA or the DNA of modification or non-modification, comprises strand and double-stranded region.

" variant " refers to be different from reference to polynucleotide or polypeptide but kept the polynucleotide or the polypeptide of fundamental characteristics.The nucleotide sequence of general polynucleotide variant is with different with reference to polynucleotide.The variation of variant nucleotide sequence may change or not change the amino acid sequence of polypeptide by the reference polynucleotide encoding.As described below, Nucleotide changes may cause amino acid whose displacement, interpolation, disappearance, fusion and brachymemma in the canonical sequence encoded polypeptides.The aminoacid sequence of general polypeptide variants is with different with reference to polypeptide.Usually, difference is limited, and therefore the sequence with reference to polypeptide and variant is extremely proximate on the whole, is identical in many zones.Variant and one or more displacements, the interpolation of any array configuration, the difference of disappearance can be arranged on aminoacid sequence with reference to polypeptide.Displacement or the amino-acid residue that inserts can yes or no genetic code amino acids coding.Polynucleotide or variant polypeptides can be naturally occurring, as allelic variant, or the non-natural existence.Polynucleotide that non-natural exists and polypeptide variants can be by induced-mutation technique or directly synthetic preparations.

" identity " is the dependency between two or more peptide sequences or two or more polynucleotide sequences as known in the art, as the case may be, can relatively come to determine by sequence.In this area, " identity " also refers to the degree that sequence between polypeptide or the polynucleotide sequence is relevant, and as the case may be, the coupling of chain that can be by these sequences is determined." identity " can use known method to calculate easily, includes but not limited to that (A.M. edits for " calculating molecular biology ", Lesk, Oxford University Press, New York, 1988; " biocomputer: information and genome plan " Smith, D.W. edits, Academic Press, New York, 1993; " Computer Analysis of sequence data " I part, Griffin, A.M. and Griffin, H.G. edits, HumanaPress, New Jersey, 1994; " sequential analysis in the molecular biology " von Heine, G., Academic Press, 1987; " sequence analysis primer " Gribskov, M. and Devereux, J. edits, M Stockton Press, New York, 1991; And Carillo, H. and Lipman, D., " SIAM J.Applied Math " 48:1073 (1988).The method of measuring identity is designed to provide the maximum match that detects between the sequence.And, measure the method for identity and in the obtainable computer program of the public, encode.The computer program means that is used for measuring two kinds of identity between the sequence includes but not limited to the GAP program (Devereux in the GCG routine package, " nucleic acids research " 12 (1) such as J.: 387 (1984), BLASTP, BLASTN (Altschul, " molecular biology magazine " 215:403-410 such as S.F. (1990) and FASTA (Pearson and Lipman " PNAS " 85:2444-2448 (1988).Blast program family can obtain from NCBI and other source (" BLAST handbook Altschul, S. etc., NCBI NLM NIH Bethesda, MD 20894; Altschul, " molecular biology magazine " 215:403-410 such as S. (1990).Famous Smith Waterman algorithm also can be used to measure identity.

The preferred algorithm that uses is FASTA.The preferred parameter that uses this algorithm to carry out polypeptide or polynucleotide sequence comparison comprises following:

Breach compensation: 12

Breach extends compensation: 4

Individual character size: 2, maximum 6

The preferred parameter that carries out the peptide sequence comparison with other method comprises following:

1) algorithm: Needleman and Wunsch, " molecular biology magazine " 48:443-453 (1970)

Comparator matrix: BLOSSUM62 sees Henikoff and Henikoff, " PNAS " 89:10915-10919 (1992)

Breach compensation: 12

Notch length compensation: 4

Use the program of these parameters can be from Genetics Computer Group, Madison WI obtains with the form of " breach " program.Above-mentioned parameter is a polypeptide default parameter (uncompensated to terminal breach) relatively.

The preferred parameter of polynucleotide sequence comparison comprises following:

1) algorithm: Needleman and Wunsch, " molecular biology magazine " 48:443-453 (1970)

Comparator matrix: coupling=+ 10, do not match=0

Breach compensation: 50

Notch length compensation: 3

Source: Genetics Computer Group, " breach " program of Madison WI.

Use the program of these parameters can be from Genetics Computer Group, Madison WI obtains with the form of " breach " program.Above-mentioned parameter is polynucleotide default parameters relatively.

For example, polynucleotide sequence of the present invention can be identical with the canonical sequence of SEQ ID NO:1, and promptly identity is 100%, perhaps compares the Nucleotide that comprises some amount with canonical sequence and change.This change can be selected from least one nucleotide deletion, displacement (comprising conversion and transversion) or insert, wherein said change can occur in reference to any position between 5 of polynucleotide sequence ' or 3 ' terminal or two ends, be dispersed in respectively between the Nucleotide of canonical sequence, perhaps be dispersed in the canonical sequence with one or more contigs.The quantity that Nucleotide changes is following to be determined: the total nucleotide number among the SEQ ID NO:1 multiply by corresponding identity percentage ratio (divided by 100), then its result is reduced from the total nucleotide number of SEQ ID NO:1, perhaps:

n_n≤x_n-(x_n·y)

N wherein_nBe the number that Nucleotide changes, x_nBe the Nucleotide sum among the SEQ ID NO:1, y is 0.70 to 70%, and to 80% being 0.80, to 85% being 0.85, to 90% being 0.90, to 95% being 0.95, the rest may be inferred, wherein as x_nWith the result of y be not integer, then it is rounded up and gets nearest integer, again from x_nMiddle subduction.The change of the polynucleotide of the polypeptide of coding SEQ ID NO:2 may produce nonsense, missense or phase shift mutation in this encoding sequence, the polypeptide of the polynucleotide encoding after therefore changing can change.

Similarly, peptide sequence of the present invention can be identical with the canonical sequence of SEQ ID NO:2, and promptly identity is 100%, perhaps compare the amino acid change that comprises some amount with canonical sequence, so identity is less than 100%.This change can be selected from least one aminoacid deletion, displacement (comprising conservative and non-conservative substitution) or insert, wherein said change can occur in reference to any position between the amino of peptide sequence or C-terminal or two ends, be dispersed in respectively between the amino acid of canonical sequence, perhaps be dispersed in the canonical sequence with one or more contigs.The quantity of identity percentage ratio one timing amino acid change is following to be determined: the total amino acid number among the SEQ IDNO:2 multiply by corresponding identity percentage ratio (divided by 100), then its result is reduced from the total amino acid number of SEQ ID NO:2, perhaps:

n_a≤x_a-(x_a·y)

N wherein_aBe amino acid change quantity, x_aIf be the total amino acid number of SEQ ID NO:2, the y value was 0.70 at 70% o'clock, was 0.80 at 80% o'clock, was 0.85 at 85% o'clock, and the rest may be inferred, wherein x_aWith the result of y be not integer, then it is rounded up and gets nearest integer, again from x_aMiddle subduction.

" homologue " is the generic term of this area, and expression has the highly polynucleotide or the peptide sequence of serial correlation with target sequence.This dependency can be come quantitatively by determining identity and/or similarity degree between the above sequence to be compared.The term that belongs in this generic term scope is " directly to a homologue " and " symbiosis homologue ", the former is the polynucleotide or the polypeptide of the function equivalent of polynucleotide or polypeptide in other species, and the latter is meant the function similar sequence when considering in same species.

Embodiment

Embodiment 1: the real-time RT-PCR analysis

Real-time RT-PCR (U.Gibson, 1996.Genome Research:6,996) is used for the abundance of comparison from candidate antigens mRNA transcript in a plurality of patients' corresponding tumour and the normal colonic tissue.In addition, the mRNA level of candidate gene is also estimated by this method in a series of healthy tissuess.

Normal and the total RNA of canceration colon uses TriPure reagent (Boehringer) by extracting in the quick-frozen biopsy samples.The total RNA of healthy tissues is available from InVitrogen or use TriPure reagent by extracting in the quick-frozen biopsy samples.The DNA enzyme is handled the back and is used oligo-dT magnetic beads (Dynal) purifying PolyA+mRNA from total RNA.(VersaFluor BioRad) carries out the quantitative of mRNA by spectrofluorimetry to use SybrII dyestuff (MolecularProbes).The primer of PCR in real time amplification adopts Perkin-Elmer Primer Express software to use the default selection design of TaqMan amplification condition.

Real time reaction is made up according to standard pcr, and the 2ng purified mRNA is used in each reaction.Adding SybrI dyestuff (Molecular Probes) to final extent of dilution when detecting in real time is 1/75000.Amplification (40 circulations) and detection in real time use conventional instrument setting to carry out in Perkin-Elmer BiosystemsPE7700 system.The Ct value is used PE7700 sequencing computed in software.Obtain each patient's two Ct value: tumour Ct (CtT) and corresponding normal colon C t (CtN).Ct value and target template copy number that PCR in real time obtains are logarithm-linear dependences.Because the efficient of pcr amplification and theoretical amplification efficiency are approaching under the normal experiment condition, can estimate relative transcriptional level (being that mRNA crosses the expression multiple in the tumour) in two kinds of tissues by 2 (CtN-CtT).Biopsy samples to 24 patients carries out real-time PCR reactions.The mRNA that calculates each patient crosses expression level.Cross expression level and patient by the antigenic average mRNA of this data set calculated candidate then and cross the ratio of expressing candidate antigens.Actin muscle stdn (ratio) in the relative same sample of single value, as shown in Figure 1.This value be 1 be equivalent to identical with the Actin muscle expression level.The result illustrates with logarithmic scale.

48 healthy tissues samples representing 28 kinds of different tissues are also by measuring with quadrat method.The value of the Actin muscle that obtains in the Ct value of candidate antigens and the homologue's sample relatively.The result is shown in Fig. 2 with respect to the Actin muscle stdn.

PCR in real time result in colorectal carcinoma/normal colon sample sums up

CASB619 crosses the patient (%) of expression in the colorectal carcinoma	The average expression level (doubly) of crossing in the colorectal carcinoma
		16/24(67％)	208

Conclusion: with respect to contiguous normal colon, CASB619 crosses in 67% colorectal carcinoma sample and expresses, and mean level (ML) is nearly 200 times.Expression in the healthy tissues is limited to other digestive tube tissue and main tracheae and testis.

Embodiment 2:DNA microarray

Dna microarray is used for checking the mRNA expression map of a large amount of genes in a plurality of samples.This information is used for remedying the data that PCR in real time obtains, and the independent tolerance of gene expression dose in tumour and the healthy tissues is provided.

The example that produces the dna microarray technology at present comprises 1) Affymetrix " gene chip (GeneChip) " array, wherein oligonucleotide uses the inorganic graphic-arts technique of luminous energy (photolithographicprocess) at the chip surface Synthetic 2) DNA spot technology, wherein a small amount of dna solution is fixed on solid phase surface (as glass) then by the robot deposition.Under two kinds of situations, chip is all with the cDNA that extracts in destination organization (as healthy tissues, tumour etc.) or cRNA hybridization and with radioactivity or with the fluorescent reporter molecule mark.The material of mark and chip hybridization, the amount that is combined in the probe of each sequence on the chip uses special scanner to determine.Experiment can use single fluorescent reporter molecule (or radioactivity) to carry out, and perhaps can carry out with two kinds of reporter molecules.Under a kind of situation in back, each in two kinds of samples is all with a kind of reporter molecules mark.Two kinds of mark samples compete then with the DNA chip on sequence hybridization.Determine the ratio of two kinds of fluorescent signals of each sequence on the chip.This ratio is used for calculating the relative abundance of transcript in two kinds of samples.Detailed method can be available from multiple source, comprise " dna microarray: practical approach; SchenaM.Oxford University Press 1999 " and internet (http://cmgm.stanford.edu/pbrown/protocols/index.html, http://arrayit.com/DNA-Microarray-Protocols/) and particular Seller (as Affymetrix)

Embodiment 3

The EST collection of illustrative plates

The additional approach that experiment antigen tissue expression is identified is to survey people " expressed sequence tag (EST) " database.EST is the small segment by the cDNA of the multiple mRNA preparation of particular organization or clone extraction.These databases provide at present from hundreds of cDNA organizes the library to comprise a large amount of EST (106) of all kinds of tumor tissues and morbid state.By gopher (Blast), carry out the relatively retrieval of CASB616 sequence, so that further explore tissue expression.

The EST of CASB619 distributes

DbEST go into to hide number	????ATGI?Iib?ID	Describe
			NCBI：1202616	????937	NCI_CGAP_Co2 (colon fine hair)
NCBI：1202659	????937	NCI_CGAP_Co2 (colon fine hair)
			NCBI：1208269	????935	NCI_CGAP_Pr4.1 (vegetation in the prostatic epithelium)
NCBI：1152744	????888	NCI_CGAP_Pr6 (vegetation in the prostatic epithelium)
			NCBI：157532	????910	NCI_CGAP_Pr22 (normal prostatic)
NCBI：1178873	????882	NCI_CGAP_Co3 (colorectal carcinomas of 12 merging)
			NCBI：1040601	????628	Testis NHT
NCBI：1056221	????628	Testis NHT
			NCBI：1298131	????910	NCI_CGAP_Pr22 (normal prostatic)
NCBI：976517	????715	Epididymis
			NCBI：1618753	????417	Parathyroid adenoma
NCBI：1737305	????895	NCI_CGAP_Br2 (breast cancer tissue of merging)
			NCBI：1738600	????895	NCI_CGAP_Br2 (breast cancer tissue of merging)
NCBI：1767617	????628	Testis NHT
			NCBI：1889992	????424	OX-heart
NCBI：1907671	????628	Testis NHT
			NCBI：2074937	????1076	NCI_CGAP_Lu5?View?stats
NCBI：2147596	????1461	NCI_CGAP_Ut1
			NCBI：2147632	????1461	NCI_CGAP_Ut1
NCBI：2308815	????1462	NCI_CGAP_Ut2
			NCBI：2441896	????1410	NCI_CGAP_Pr28
NCBI：2447648	????1463	NCI_CGAP_Ut3
			NCBI：2381271	????1461	NCI_CGAP_Ut1
NCBI：2582781	????1461	NCI_CGAP_Ut1
			NCBI：2583713	????1461	NCI_CGAP_Ut1
NCBI：2585593	????1410	NCI_CGAP_Pr28
			NCBI：2587322	????1463	NCI_CGAP_Ut3
NCBI：2601136	????1461	NCI_CGAP_Ut1
			NCBI：2824305	????1463	NCI_CGAP_Ut3
NCBI：3007221	????1461	NCI_CGAP_Ut1
			NCBI：3078572	????2301	NCI_CGAP_Pt1
NCBI：3086989	????1461	NCI_CGAP_Ut1
			NCBI：3087235	????1461	NCI_CGAP_Ut1
NCBI：3045696	????1661	NCI_CGAP_Lu19
			NCBI：3218161	????2467	NCI_CGAP_Co20
NCBI：3291383	????2508	NCI_CGAP_Sub3
			NCBI：3028913	????2107	BT130
NCBI：3655706	????1447	NCI_CGAP_Co14
			NCBI：2553046	????1728	Soares_Dieckgraefe_ colon _ NHCD
NCBI：979345	????781	Adenomyoma
			NCBI：3289738	????2508	NCI_CGAP_Sub3
NCBI：614598	????464	？
			NCBI：978076	????781	Adenomyoma
NCBI：979060	????781	Adenomyoma
			NCBI：1616799	????417	Parathyroid adenoma

NCBI：978445

????781

Adenomyoma

In a word, the 93%EST with the CASB619 coupling derives from tumor tissues, ox tissue or normal reproductive organ.This shows that this gene is more common in the expression of this tissue.

Embodiment 4

The RNA-DNA engram analysis

Mixed rumour and corresponding normal colon cDNA by Advantage PCR (on seeing) amplification limited amount.The messenger RNA(mRNA) of a plurality of healthy tissuess also uses identical method amplification.CDNA (1 μ g) electrophoresis on 1.2% sepharose of amplification, and transfer on the nylon membrane.This film and the probe hybridization (AlkPhos DirectSystem) of using candidate TAA cDNA produced in fragments.RNA-DNA analyzes the information that transcript abundance in transcript size, splice variant existence and tumour and the healthy tissues is provided.

Embodiment 5

Rna blot analysis

The RNA trace uses 1 μ g polyA+mRNA to prepare according to standard method.The radioactivity probe uses Ready-to-Go system (Pharmacia) preparation.

Embodiment 6

Identify full length cDNA sequence

Colorectal carcinoma cDNA library uses λ Zap II system (Stratagene) to be made up by 5 microgram polyA+mRNA.According to the method that provides, but use SuperscriptII (LifeTechnologies) in the reverse transcription step.Make up library that Oligo-dT causes and that cause at random.Be coated with about 1.5 * 10 when screening each library⁶Phagus liber.Have a liking for bacterial plaque and transfer on the nylon leaching film, with cDNA probe hybridization with AlkPhos Direct mark.Positive bacteriophage passes through chemiluminescence detection.By downcutting positive bacteriophage on the agar plate, wash-out in 500 microlitre SM damping fluids confirms by gene specific PCR.The phage of wash-out is transformed into strand M13 phage by excision in the body.Phage is transformed into double-stranded plasmid DNA by ehec infection then.The bacterium bed board that infects carries out second with the cDNA probe then and takes turns screening.Check order by positive bacteria clone purification plasmid DNA and to two strands.When full-length gene can not directly be obtained by the cDNA library, the sequence of disappearance use the RACE technical point from (Marathon Kit, ClonTech).This method depends on reverse transcription mRNA and becomes double-stranded cDNA, and joint is connected to the end of cDNA, and uses the increase end of required cDNA of gene-specific primer and joint oligonucleotide.Marathon PCR product cloning is advanced in the plasmid (pCRII-TOPO) and order-checking.

The sequence (SEQ ID NO:1) that obtains has one 1013 amino acid whose supposition open reading frame (SEQ ID NO:2).To the protein sequence of inferring carried out the cellular localization prediction and calculation (PSORT:http: //psort.nibb.ac.jp/and TopPred:http: //www.biokemi.su.se/～sever/toppred2/toppred_source.html).As if CASB619 have peptide signal and one to three and stride film district (low confidence prediction).Do not find other primitive or structural domain.

Embodiment 7:

7.1 the expression of tumour specific antigen and purifying

Expression in microorganism host or in-vitro transcription/translation system are used to produce the antigen of the present invention as the vaccine purpose, or generation is identified the fast purifying and the generation of the antibody that natural expressing protein is required or is further purified required protein fragments or intact proteins by immunohistochemistry.

Recombinant protein can be expressed in two kinds of microorganism host intestinal bacteria and yeast (as yeast saccharomyces cerevisiae or pichia pastoris phaff).This can select to produce for specific antigen the expression system of advantageous feature.Generally, recombinant antigen will be at expression in escherichia coli, and reagent albumen is expressed in yeast.

Expression strategy at first relates to the primary structure of recombinant antigen.Generally, express fusion partner (EFP) and be placed in N-terminal,, can also comprise zone, the Immune Fusion mating partner (IFP) that is used for regulating antigenic immunogen characteristic at N-terminal to improve expression level.In addition, the affine usefulness fusion partner (AFP) of helping be further purified is placed in C-terminal.

When having obtained recombinant bacterial strain, identify recombinant products by the evaluation expression level, by analyzing the proteic further solvability of behavior prediction of crude extract.

In suitable growth medium the growth and induce expression of recombinant proteins after, analyze whole extracts by SDS-PAGE.Recombinant protein develops the color on the dyeing gel, uses specific antibody to identify by western blot analysis.

The comparison of different types antigen expressed can be selected most promising candidate, and it will be used to be further purified and immunoassay.

Purification scheme is used the ordinary method when having the affine tail of His in the recombinant protein.In typical experiment, filter smudge cells, sample on the cell-free extract is kept the ionic metal affinity chromatography (IMAC of recombinant protein to specificity; Ni++NTA is from Qiagen) on.

The albumen that keeps is by 0-500mM imidazoles gradient (may the have denaturing agent) wash-out in the phosphoric acid buffer.

7.2 Antibody Preparation and immunohistochemistry

A small amount of albumen of purifying relatively can be used to produce immune instrument, so that

A) detect expression by immunohistochemistry normal or the cancerous tissue section;

B) albumen (ELISA/ Western blot) is expressed and is followed the tracks of in detection in purge process; Or

C) identify/quantitative purifying protein (ELISA).

7.2.1 polyclonal antibody:

Immunity

Pass through 2-3 rabbit ofintramuscular approach immunity 3 times with 100 μ g albumen, in each 3 weeks at interval, albumen is formulated among the adjuvant 3D-MPL/QS21.Each 3 week of immunity the back blood samplings, use as the albumen of envelope antigen and measure antibody titers in the serum by ELISA according to standard method.

ELISA

96 hole micro plates (maxisorb Nunc) are spent the night with 4 ℃ of bags of 5 μ g albumen.37 ℃ after saturated 1 hour, add the serial dilutions (1/10 beginning) of rabbit anteserum, 37 ℃ of 1H 30 with PBS NCS 1%.After thePBS Tween washing 3 times, add anti-rabbit biotinylation antiserum(antisera) (Amersham) (1/5000).Washing is dull and stereotyped, adds peroxidase link coupled streptavidin (1/5000), 37 ℃ 30 minutes.After the washing, add 50 μ l TMB (BioRad) 7 minutes, use the sulfuric acid termination reaction then.Measure OD at 450nm, calculate the mid point extent of dilution by SoftmaxPro.

7.2.2 monoclonal antibody:

Immunity

With 5 μ g purifying proteins with 5 BALB/c mouse of the interval in 3weeks immunity 3 times.1 week got blood after 14 days and the 3rd time after the 2nd time.Measure serum as envelope antigen by Elisa with purifying protein.According to these results (mid point extent of dilution＞10000), select 1 mouse to be used for merging.

Fusion/HAT selects

Use PEG 40% and DMSO 5% that splenocyte and SP2/0 myelomatosis are merged according to standard method.Cell inoculation 96 orifice plates, every hole 2.5 * 10⁴-10⁵Cell is selected resistance clone on the HAT substratum.The content of specific antibody is positive as the result in the supernatant of detection hybridoma, hybridoma is carried out 2 take turns limiting dilution.2 take turns screening after, select 3 hybridomas to be used for ascites and generate.

7.2.3 immunohistochemistry

After obtaining antibody, in normal and cancerous issue section, carry out immunostaining, so that determine:

In ◇ canceration and the healthy tissues antigen levels of the present invention or

The ratio of certain types of cancer of ◇ antigen expressed

If other cancer types of ◇ is antigen expressed also

◇ comprises the ratio of antigenic cell in cancerous issue

The tissue sample preparation

After the incision, tissue sample places the cork dish of OCH compound, in quick-frozen in the iso-pentane of deep refrigeration in liquid nitrogen (one 160 ℃).Sample retention is stand-by at-70 ℃.In freezer compartment (20 ℃ ,-30 ℃), cut out 7-10 μ m section.

Dyeing

Tissue slice is drying at room temperature 5 minutes, and under the room temperature in acetone fixedly l0 minute, after drying is saturated with PBS 0.5%BSA 5% serum.After following 30 minutes of the room temperature, use antigen-specific antibodies directly or indirectly to dye.The substantive dyeing specificity is better, but a little less than the dyeing, and dyeing dyeing is stronger indirectly, but specificity is relatively poor.

7.3 analyze at the antigenic people's cellullar immunologic response of the present invention

The antigenic immune association of the present invention can be estimated by external initiation human T-cell.All T lymphocyte series and dendritic cell derive from healthy donors (preferred HLA-A2 hypotype) PBMC (peripheral blood lymphocytes).The HLA-A2.1/Kb transgenic mice also is used to screen the HLA-A2.1 peptide.

Produce and keep newfound antigen-specific CD8+T clone by stimulated in vitro weekly.Cd8 cell system at the lytic activity of antigen and antigen derived peptide and

IFN produces and uses standard method to measure.

Use two kinds of strategies to produce antigen-specific CD8+T cells: based on the approach of peptide with based on the approach of complete genome.Two kinds of approach all need the antigenic full-length cDNA of new discovery, are cloned in the suitable transfer system with accurate frame, perhaps are used for predicting the HLA peptide binding sequence.

Approach based on peptide

HLA-A2 peptide binding sequence Parker in romote antiquity algorithm (Parker, K.C., M.A.Bednarek, and J.E.Coligan.1994 is independent of with potential HLA-A2 binding peptide fractionated scheme based on independent peptide side chain.Journal of Immunology, 152:163 and http://bimas.dcrt.nih.gov/molbio/hla_bind/) or Rammensee method (Rammensee, Friede, Stevanovic, MHC part and peptide primitive: table 1, immunogenetics 41,178-228,1995; Rammensee, Bachmann, Stevanovic:MHC part and peptide primitive.Landes Bioscience 1997, and http:134.2.96.221/scriptsh/laserver.dll/home.htm).In HLA-A2.1/Kb transgene mouse model (Vitiello et al), screen peptide then.

A) Yu Ce epi-position in conjunction with HLA A0201 locus

A.1) HLA-A^*0,201 nine aggressiveness

The position	??1??2??3??4??5??6??7??8??9	Rammens ee score	Parker score °	??SEQID ????NO
					??848	??F??L??W??E??S??A??A??A??C		??777.681	????68
??24	??R??L??W??R??L??L??L??W??A		??521.615	????5
					??761	??S??L??A??D??R??L??I??G??V	????30	??655.875	????6
??893	??S??L??P??E??Q??R??V??T??I	????26		????7
					??886	??K??L??C??S??G??G??I??S??L	????25		????8
??853	??A??A??A??C??P??L??C??S??V	????25		????9
					??674	??S??A??L??A??N??T??V??T??L	????24		????10
??499	??F??V??F??E??T??L??C??S??V	????24	??976.762	????11
					??129	??E??L??P??H??G??F??A??S??L	????23		????12
??973	??L??I??F??T??S??K??K??S??L	????22		????13
					??936	??K??L??E??Y??K??Y??S??K??L	????22		????14
??903	??K??T??I??D??F??W??L??K??V	????22		????15
					??860	??S??V??A??D??Y??H??A??I??V	????22		????16
??830	??L??L??L??P??G??T??C??S??D	????22		????17
					??675	??A??L??A??N??T??V??T??L??A	????22		????18
??503	??T??L??C??S??V??N??C??E??L	????22		????19
					??169	??N??T??D??E??C??T??A??T??L	????22		????20
??81	??S??L??P??D??P??V??K??G??T	????22		????21
					??980	??S??L??F??G??K??I??K??S??F	????21		????22
??918	??C??T??A??I??L??L??T??V??L	????21		????23
					??867	??I??V??S??S??C??V??A??G??I	????21		????24
??710	??K??M??S??V??C??T??D??N??V	????21		????25
					??259	??V??L??V??R??N??I??A??I??T	????21		????26
??234	??E??L??N??R??G??N??N??V??L	????21		????27
					??175	??A??T??L??M??Y??A??V??N??L	????21		????28
??70	??V??A??V??P??H??T??P??G??L	????21		????29
					??24	??R??L??W??R??L??L??L??W??A	????21		????30
??914	??S??A??G??T??C??T??A??I??L	????20		????31
					??891	??G??I??S??L??P??E??Q??R??V	????20		????32
??824	??K??T??V??P??G??S??L??L??L	????20		????33
					??765	??R??L??I??G??V??T??T??D??M	????20		????34

??681	??T??L??A??G??G??P??S??F??T	????20		????35
					??539	??Y??I??I??E??E??N??T??T??T	????20		????36
??264	??I??A??I??T??G??V??A??Y??T	????20		????37
					??38	??V??T??Q??G??T??G??P??E??L	????20		????38

The molecule that ° contains this subsequence is estimated to dissociate between half

A.2) HLA A02_01 ten aggressiveness

The position	?1??2??3??4??5??6??7??8??9??10	Rammens ee score	Parker score °	??SEQID ????NO
					??980	?S??L??F??G??K??I??K??S??F??T		??151.648	????39
??866	?A??I??V??S??S??C??V??A??G??I	????25		????40
					??852	?S??A??A??A??C??P??L??C??S??V	????24		????41
??786	?H??L??E??S??L??G??I??P??D??V	????24		????42
					??571	?K??I??Y??S??I??N??V??T??N??V	????24	??246.353	????43
??761	?S??L??A??D??R??L??I??G??V??T	????23		????44
					??626	?I??L??K??A??H??Q??P??Y??G??V	????23		????5
??485	?V??M??A??D??T??E??N??K??E??V	????23	??350.117	????46
					??29	?L??L??W??A??G??T??A??F??Q??V	????23	??5691.997	????47
??916	?G??T??C??T??A??I??L??L??T??V	????22		????48
					??778	?I??T??S??P??A??E??L??F??H??L	????22		????49
??766	?L??I??G??V??T??T??D??M??T??L	????22		????50
					??428	?T??L??P??T??N??M??E??T??T??V	????22		????51
??350	?L??M??Y??K??W??A??K??P??K??I	????22		????52
					??972	?D??L??I??F??T??S??K??K??S??L	????21		????53
??692	?G??L??K??Y??F??H??H??F??T??L	????21		????54
					??644	?G??T??K??N??N??K??I??H??S??L	????21		????55
??465	?S??D??N??D??F??M??I??L??T??L	????21		????56
					??260	?L??V??R??N??I??A??I??T??G??V	????21		????57
??77	?G??L??C??T??S??L??P??D??P??V	????21		????58
					??949	?T??L??K??D??C??D??L??P??A??A	????20		????59
??795	?V??I??F??F??Y??R??S??N??D??V	????20		????60
					??733	?S??I??T??A??Y??V??C??Q??A??V	????20		????61
??712	?S??V??C??T??D??N??V??T??D??L	????20		????62
					??702	?S??L??C??G??N??Q??G??R??K??M	????20		????63
??432	?N??M??E??T??T??V??L??S??G??I	????20		????64
					??263	?N??I??A??I??T??G??V??A??Y??T	????20		????65
??121	?G??I??R??F??D??E??W??D??E??L	????20		????66

The molecule that ° contains this subsequence is estimated to dissociate between half

HLA_A0205

The position	?1??2??3??4??5??6??7??8??9	Parker score °	??SEQID ????NO
				?499	?F??V??F??E??T??L??C??S??V	?216	????11

HLA?A0203

The position	??1??2??3??4??5??6??7??8??9??10	Rammens ee score	??SEQID ????NO
				?846	??F??H??F??L??W??E??S??A??A??A	??27	????67

In brief, with the HLA-A2 peptide immune transgenic mouse of adjuvant preparation, can not induce the peptide that CD8 replys (be defined as effective cleavage of peptide burst process from body homology splenocyte) in robot system, further to handle.

People's dendritic cell (according to cultivations such as Romani) are with the peptide burst process and be used to stimulate the T cell (Facs) of CD8-sorting.After stimulating several weeks, cd8 cell system at first tests going up from body homology BLCL (EBV-B transformation cell lines) of peptide burst process.In order to process in the accurate body of proving conclusively peptide, test will be gone up at the tumour cell (LnCaP of HLA-A2 transfection, Skov3 or CAMA tumour cell) of cDNA transfection by cd8 cell system.

Approach based on full gene

The dendritic cell that infect with inoblast, recombinant poxvirus (Kim etc.) or the adenovirus (Butterfield etc.) of the B7.1 transfection of the dendritic cell of particle gun transfection, retrovirus transduction cause and stimulate CD8+T clone.Virus infected cell is the antigen-presenting peptide very effectively, because antigen is with high level expression, but can only use once, to avoid the hypertrophy of viral T clone.

After alternately stimulating, CD8+ clone is fastened test at the tumour cell of aforesaid cDNA transfection.Determine that peptide specific and identity are to confirm immune validity.

Reference

Vitiello etc. (L.Sherman), The Journal of Experimental Medicine 1991,173:1007-1015.

Romani etc., The Journal of Experimental Medicine, 1994,180:83-93.

Kim etc., immunotherapy, 1997,20:276-286.

Butterfield etc., Journal of Immunology, 1998,161:5607-5613.

All publications and the reference quoted in the application's book include but not limited to patent and patent application, here all are incorporated herein by reference in full, all list in full at this especially and individually separately as them.SEQ ID NO：1atggctgagcctgggcacagccaccatctctccgccagagtcaggggaagaactgagaggcgcataccccggctgtggcggctgctgctctgggctgggaccgccttccaggtgacccagggaacgggaccggagcttcatgcctgcaaagagtctgagtaccactatgagtacacggcgtgtgacagcacgggttccaggtggagggtcgccgtgccgcataccccgggcctgtgcaccagcctgcctgaccccgtcaagggcaccgagtgctccttctcctgcaacgccggggagtttctggatatgaaggaccagtcatgtaagccatgcgctgagggccgctactccctcggcacaggcattcggtttgatgagtgggatgagctgccccatggctttgccagcctctcagccaacatggagctggatgacagtgctgctgagtccaccgggaactgtacttcgtccaagtgggttccccggggcgactacatcgcctccaacacggacgaatgcacagccacactgatgtacgccgtcaacctgaagcaatctggcaccgttaacttcgaatactactatccagactccagcatcatctttgagtttttcgttcagaatgaccagtgccagcccaatgcagatgactccaggtggatgaagaccacagagaaaggatgggaattccacagtgtggagctaaatcgaggcaataatgtcctctattggagaaccacagccttctcagtatggaccaaagtacccaagcctgtgctggtgagaaacattgccataacaggggtggcctacacttcagaatgcttcccctgcaaacctggcacgtatgcagacaagcagggctcctctttctgcaaactttgcccagccaactcttattcaaataaaggagaaacttcttgccaccagtgtgaccctgacaaatactcagagaaaggatcttcttcctgtaacgtgcgcccagcttgcacagacaaagattatttctacacacacacggcctgcgatgccaacggagagacacaactcatgtacaaatgggccaagccgaaaatctgtagcgaggaccttgagggggcagtgaagctgcctgcctctggtgtgaagacccactgcccaccctgcaacccaggcttcttcaaaaccaacaacagcacctgccagccctgcccatatggttcctactccaatggctcagactgtacccgctgccctgcagggactgaacctgctgtgggatttgaatacaaatggtggaacacgctgcccacaaacatggaaacgaccgttctcagtgggatcaacttcgagtacaagggcatgacaggctgggaggtggctggtgatcacatttacacagctgctggagcctcagacaatgacttcatgattctcactctggttgtgccaggatttagacctccgcagtcggtgatggcagacacagagaataaagaggtggccagaatcacatttgtctttgagaccctctgttctgtgaactgtgagctctacttcatggtgggtgtgaattctaggaccaacactcctgtggagacgtggaaaggttccaaaggcaaacagtcctatacctacatcattgaggagaacactaccacgagcttcacctgggccttccagaggaccacttttcatgaggcaagcaggaagtacaccaatgacgttgccaagatctactccatcaatgtcaccaatgttatgaatggcgtggcctcctactgccgtccctgtgccctagaagcctctgatgtgggctcctcctgcacctcttgtcctgctggttactatattgaccgagattcaggaacctgccactcctgcccccctaacacaattctgaaagcccaccagccttatggtgtccaggcctgtgtgccctgtggtccagggaccaagaacaacaagatccactctctgtgctacaatgattgcaccttctcacgcaacactccaaccaggactttcaactacaacttctccgctttggcaaacaccgtcactcttgctggagggccaagcttcacttccaaagggttgaaatacttccatcactttaccctcagtctctgtggaaaccagggtaggaaaatgtctgtgtgcaccgacaatgtcactgacctccggattcctgagggtgagtcagggttctccaaatctatcacagcctacgtctgccaggcagtcatcatccccccagaggtgacaggctacaaggccggggtttcctcacagcctgtcagccttgctgatcgacttattggggtgacaacagatatgactctggatggaatcacctccccagctgaacttttccacctggagtccttgggaataccggacgtgatcttcttttataggtccaatgatgtgacccagtcctgcagttctgggagatcaaccaccatccgcgtcaggtgcagtccacagaaaactgtccctggaagtttgctgctgccaggaacgtgctcagatgggacctgtgatggctgcaacttccacttcctgtgggagagcgcggctgcttgcccgctctgctcagtggctgactaccatgctatcgtcagcagctgtgtggctgggatccagaagactacttacgtgtggcgagaacccaagctatgctctggtggcatttctctgcctgagcagagagtcaccatctgcaaaaccatagatttctggctgaaagtgggcatctctgcaggcacctgtactgccatcctgctcaccgtcttgacctgctacttttggaaaaagaatcaaaaactagagtacaagtactccaagctggtgatgaatgctactctcaaggactgtgacctgccagcagctgacagctgcgccatcatggaaggcgaggatgtagaggacgacctcatctttaccagcaagaagtcactctttgggaagatcaaatcatttacctccaagaggactcctgatggatttgactcagtgccgctgaagacatcctcaggaggcccagacatggacctgtgaGAGGCACTGCCTGCCTCACCTGCCTCCTCACCTTGCATAGCACCTTTGCAAGCCTGCGGCGATTTGGGTGCCAGCATCCTGCAACACCCACTGCTGGAAATCTCTTCATTGTGGCCTTATCAGATGTTTGAATTTCAGATCTTTTTTTATAGAGTACCCAAACCCTCCTTTCTGCTTGCCTCAAACCTGCCAAATATACCCACACTTTGTTTGTAAAAaaaAAAAAAAAAAAAAAAAASEQ ID NO：2MAEPGHSHHLSARVRGRTERRIPRLWRLLLWAGTAFQVTQGTGPELHACKESEYHYEYTACDSTGSRWRVAVPHTPGLCTSLPDPVKGTECSFSCNAGEFLDMKDQSCKPCAEGRYSLGTGIRFDEWDELPHGFASLSANMELDDSAAESTGNCTSSKWVPRGDYIASNTDECTATLMYAVNLKQSGTVNFEYYYPDSSIIFEFFVQNDQCQPNADDSRWMKTTEKGWEFHSVELNRGNNVLYWRTTAFSVWTKVPKPVLVRNIAITGVAYTSECFPCKPGTYADKQGSSFCKLCPANSYSNKGETSCHQCDPDKYSEKGSSSCNVRPACTDKDYFYTHTACDANGETQLMYKWAKPKICSEDLEGAVKLPASGVKTHCPPCNPGFFKTNNSTCQPCPYGSYSNGSDCTRCPAGTEPAVGFEYKWWNTLPTNMETTVLSGINFEYKGMTGWEVAGDHIYTAAGASDNDFMILTLVVPGFRPPQSVMADTENKEVARITFVFETLCSVNCELYFMVGVNSRTNTPVETWKGSKGKQSYTYIIEENTTTSFTWAFQRTTFHEASRKYTNDVAKIYSINVTNVMNGVASYCRPCALEASDVGSSCTSCPAGYYIDRDSGTCHSCPPNTILKAHQPYGVQACVPCGPGTKNNKIHSLCYNDCTFSRNTPTRTFNYNFSALANTVTLAGGPSFTSKGLKYFHHFTLSLCGNQGRKMSVCTDNVTDLRIPEGESGFSKSITAYVCQAVIIPPEVTGYKAGVSSQPVSLADRLIGVTTDMTLDGITSPAELFHLESLGIPDVIFFYRSNDVTQSCSSGRSTTIRVRCSPQKTVPGSLLLPGTCSDGTCDGCNFHFLWESAAACPLCSVADYHAIVSSCVAGIQKTTYVWREPKLCSGGISLPEQRVTICKTIDFWLKVGISAGTCTAILLTVLTCYFWKKNQKLEYKYSKLVMNATLKDCDLPAADSCAIMEGEDVEDDLIFTSKKSLFGKIKSFTSKRTPDGFDSVPLKTSSGGPDMDLSEQ ID NO：3TTTTTTAATTTACAAACAAAGTGTGGGTATATTTGGCAGGTTTGAGGCAAGCAGAAAGGAGGGTTTGGGTACTCTATAAAAAAAGATCTGAAATTCAAACATCTGATAAGGCCACAATGAAGAGATTTCCAGCAGTGGGTGTTGCAGGATGCTGGCACCCAAATCGCCGCACGTTGCAAAGGTGCTATGCAAGGTGAGGAGGCAGGTGAGGCAGGCAGTGCCTCTCACAGGTCCATGTCTGGGCCTCCTGAGGATGTCTTCAGCGGCACTGAGTCAAATCCATCAGGAGTCCTCTTGGAGGTAAATGATTTGATCTTCCCAAAGAGTGACTTCTTGCTGGTAAAGATGAGGTCGTCCTCTACATCCTCGCCTTCCATGATGGCGCAGTGTCAGCTGCTGGCAGGTCACAGTCCTTGAGAGTAGCATTCATCACCAGCTTGGAGTACTTGTACTCTAGTTTTTGATTCTTTTTCCAAAAGTAGCAGGTCAAGACGGTGAGCAGGATGGCAGTACAGGTGCCTGCAGAGATGCCCACTTTCAGCCAGAAATCTATGGTTTTGCAGATGGTGACTCTCTGCTCAGGCAGAGAAATGCCACCAGAGCATAGCTTGGGTTCTCGCCACACGTAAGTAGTCTTCTGGATCCCAGCCACACAGCTGCTGACGATAGCATGGTAGSEQ ID NO：4YHAIVSSCVAGIQKTTYVWREPKLCSGGISLPEQRVTICKTIDFWLKVGISAGTCTAILLTVLTCYFWKKNQKLEYKYSKLVMNATLKDCDLPAADTAPSWKARM

Sequence table

<110>Smithkline?Beecham?Biologicals?S.A.

＜120〉new compound

<130>BC45226

<160>68

<170>FastSEQ?for?Windows?Version?3.0

<210>1

<211>3280

<212>DNA

＜213〉people

<400>?1atggctgagc?ctgggcacag?ccaccatctc?tccgccagag?tcaggggaag?aactgagagg?????60cgcatacccc?ggctgtggcg?gctgctgctc?tgggctggga?ccgccttcca?ggtgacccag????120ggaacgggac?cggagcttca?tgcctgcaaa?gagtctgagt?accactatga?gtacacggcg????180tgtgacagca?cgggttccag?gtggagggtc?gccgtgccgc?ataccccggg?cctgtgcacc????240agcctgcctg?accccgtcaa?gggcaccgag?tgctccttct?cctgcaacgc?cggggagttt????300ctggatatga?aggaccagtc?atgtaagcca?tgcgctgagg?gccgctactc?cctcggcaca????360ggcattcggt?ttgatgagtg?ggatgagctg?ccccatggct?ttgccagcct?ctcagccaac????420atggagctgg?atgacagtgc?tgctgagtcc?accgggaact?gtacttcgtc?caagtgggtt????480ccccggggcg?actacatcgc?ctccaacacg?gacgaatgca?cagccacact?gatgtacgcc????540gtcaacctga?agcaatctgg?caccgttaac?ttcgaatact?actatccaga?ctccagcatc????600atctttgagt?ttttcgttca?gaatgaccag?tgccagccca?atgcagatga?ctccaggtgg????660atgaagacca?cagagaaagg?atgggaattc?cacagtgtgg?agctaaatcg?aggcaataat????720gtcctctatt?ggagaaccac?agccttctca?gtatggacca?aagtacccaa?gcctgtgctg????780gtgagaaaca?ttgccataac?aggggtggcc?tacacttcag?aatgcttccc?ctgcaaacct????840ggcacgtatg?cagacaagca?gggctcctct?ttctgcaaac?tttgcccagc?caactcttat????900tcaaataaag?gagaaacttc?ttgccaccag?tgtgaccctg?acaaatactc?agagaaagga????960tcttcttcct?gtaacgtgcg?cccagcttgc?acagacaaag?attatttcta?cacacacacg????1020gcctgcgatg?ccaacggaga?gacacaactc?atgtacaaat?gggccaagcc?gaaaatctgt????1080agcgaggacc?ttgagggggc?agtgaagctg?cctgcctctg?gtgtgaagac?ccactgccca????1140ccctgcaacc?caggcttctt?caaaaccaac?aacagcacct?gccagccctg?cccatatggt????1200tcctactcca?atggctcaga?ctgtacccgc?tgccctgcag?ggactgaacc?tgctgtggga????1260tttgaataca?aatggtggaa?cacgctgccc?acaaacatgg?aaacgaccgt?tctcagtggg????1320atcaacttcg?agtacaaggg?catgacaggc?tgggaggtgg?ctggtgatca?catttacaca????1380gctgctggag?cctcagacaa?tgacttcatg?attctcactc?tggttgtgcc?aggatttaga????1440cctccgcagt?cggtgatggc?agacacagag?aataaagagg?tggccagaat?cacatttgtc????1500tttgagaccc?tctgttctgt?gaactgtgag?ctctacttca?tggtgggtgt?gaattctagg????1560accaacactc?ctgtggagac?gtggaaaggt?tccaaaggca?aacagtccta?tacctacatc????1620attgaggaga?acactaccac?gagcttcacc?tgggccttcc?agaggaccac?ttttcatgag????1680gcaagcagga?agtacaccaa?tgacgttgcc?aagatctact?ccatcaatgt?caccaatgtt????1740atgaatggcg?tggcctccta?ctgccgtccc?tgtgccctag?aagcctctga?tgtgggctcc????1800tcctgcacct?cttgtcctgc?tggttactat?attgaccgag?attcaggaac?ctgccactcc????1860tgccccccta?acacaattct?gaaagcccac?cagccttatg?gtgtccaggc?ctgtgtgccc????1920tgtggtccag?ggaccaagaa?caacaagatc?cactctctgt?gctacaatga?ttgcaccttc????1980tcacgcaaca?ctccaaccag?gactttcaac?tacaacttct?ccgctttggc?aaacaccgtc????2040actcttgctg?gagggccaag?cttcacttcc?aaagggttga?aatacttcca?tcactttacc????2100ctcagtctct?gtggaaacca?gggtaggaaa?atgtctgtgt?gcaccgacaa?tgtcactgac????2160ctccggattc?ctgagggtga?gtcagggttc?tccaaatcta?tcacagccta?cgtctgccag????2220gcagtcatca?tccccccaga?ggtgacaggc?tacaaggccg?gggtttcctc?acagcctgtc????2280agccttgctg?atcgacttat?tggggtgaca?acagatatga?ctctggatgg?aatcacctcc????2340ccagctgaac?ttttccacct?ggagtccttg?ggaataccgg?acgtgatctt?cttttatagg????2400tccaatgatg?tgacccagtc?ctgcagttct?gggagatcaa?ccaccatccg?cgtcaggtgc????2460agtccacaga?aaactgtccc?tggaagtttg?ctgctgccag?gaacgtgctc?agatgggacc????2520tgtgatggct?gcaacttcca?cttcctgtgg?gagagcgcgg?ctgcttgccc?gctctgctca????2580gtggctgact?accatgctat?cgtcagcagc?tgtgtggctg?ggatccagaa?gactacttac????2640gtgtggcgag?aacccaagct?atgctctggt?ggcatttctc?tgcctgagca?gagagtcacc????2700atctgcaaaa?ccatagattt?ctggctgaaa?gtgggcatct?ctgcaggcac?ctgtactgcc????2760atcctgctca?ccgtcttgac?ctgctacttt?tggaaaaaga?atcaaaaact?agagtacaag???2820tactccaagc?tggtgatgaa?tgctactctc?aaggactgtg?acctgccagc?agctgacagc???2880tgcgccatca?tggaaggcga?ggatgtagag?gacgacctca?tctttaccag?caagaagtca???2940ctctttggga?agatcaaatc?atttacctcc?aagaggactc?ctgatggatt?tgactcagtg???3000ccgctgaaga?catcctcagg?aggcccagac?atggacctgt?gagaggcact?gcctgcctca???3060cctgcctcct?caccttgcat?agcacctttg?caagcctgcg?gcgatttggg?tgccagcatc???3120ctgcaacacc?cactgctgga?aatctcttca?ttgtggcctt?atcagatgtt?tgaatttcag???3180atcttttttt?atagagtacc?caaaccctcc?tttctgcttg?cctcaaacct?gccaaatata???3240cccacacttt?gtttgtaaaa?aaaaaaaaaa?aaaaaaaaaa?????????????????????????3280

<210>2

<211>1013

<212>PRT

＜213〉people

<400>?2Met?Ala?Glu?Pro?Gly?His?Ser?His?His?Leu?Ser?Ala?Arg?Val?Arg?Gly1????????????????5??????????????????10??????????????????15Arg?Thr?Glu?Arg?Arg?Ile?Pro?Arg?Leu?Trp?Arg?Leu?Leu?Leu?Trp?Ala

20??????????????????25??????????????????30Gly?Thr?Ala?Phe?Gln?Val?Thr?Gln?Gly?Thr?Gly?Pro?Glu?Leu?His?Ala

35??????????????????40??????????????????45Cys?Lys?Glu?Ser?Glu?Tyr?His?Tyr?Glu?Tyr?Thr?Ala?Cys?Asp?Ser?Thr

50??????????????????55??????????????????60Gly?Ser?Arg?Trp?Arg?Val?Ala?Val?Pro?His?Thr?Pro?Gly?Leu?Cys?Thr65??????????????????70??????????????????75??????????????????80Ser?Leu?Pro?Asp?Pro?Val?Lys?Gly?Thr?Glu?Cys?Ser?Phe?Ser?Cys?Asn

85??????????????????90??????????????????95Ala?Gly?Glu?Phe?Leu?Asp?Met?Lys?Asp?Gln?Ser?Cys?Lys?Pro?Cys?Ala

100?????????????????105?????????????????110Glu?Gly?Arg?Tyr?Ser?Leu?Gly?Thr?Gly?Ile?Arg?Phe?Asp?Glu?Trp?Asp

115?????????????????120?????????????????125Glu?Leu?Pro?His?Gly?Phe?Ala?Ser?Leu?Ser?Ala?Asn?Met?Glu?Leu?Asp

130?????????????????135?????????????????140Asp?Ser?Ala?Ala?Glu?Ser?Thr?Gly?Asn?Cys?Thr?Ser?Ser?Lys?Trp?Val145?????????????????150?????????????????155?????????????????160Pro?Arg?Gly?Asp?Tyr?Ile?Ala?Ser?Asn?Thr?Asp?Glu?Cys?Thr?Ala?Thr

165?????????????????170?????????????????175Leu?Met?Tyr?Ala?Val?Asn?Leu?Lys?Gln?Ser?Gly?Thr?Val?Asn?Phe?Glu

180?????????????????185?????????????????190Tyr?Tyr?Tyr?Pro?Asp?Ser?Ser?Ile?Ile?Phe?Glu?Phe?Phe?Val?Gln?Asn

195?????????????????200?????????????????205Asp?Gln?Cys?Gln?Pro?Asn?Ala?Asp?Asp?Ser?Arg?Trp?Met?Lys?Thr?Thr

210?????????????????215?????????????????220Glu?Lys?Gly?Trp?Glu?Phe?His?Ser?Val?Glu?Leu?Asn?Arg?Gly?Asn?Asn225?????????????????230?????????????????235?????????????????240Val?Leu?Tyr?Trp?Arg?Thr?Thr?Ala?Phe?Ser?Val?Trp?Thr?Lys?Val?Pro

245?????????????????250?????????????????255Lys?Pro?Val?Leu?Val?Arg?Asn?Ile?Ala?Ile?Thr?Gly?Val?Ala?Tyr?Thr

260?????????????????265?????????????????270Ser?Glu?Cys?Phe?Pro?Cys?Lys?Pro?Gly?Thr?Tyr?Ala?Asp?Lys?Gln?Gly

275?????????????????280?????????????????285Ser?Ser?Phe?Cys?Lys?Leu?Cys?Pro?Ala?Asn?Ser?Tyr?Ser?Asn?Lys?Gly

290?????????????????295?????????????????300Glu?Thr?Ser?Cys?His?Gln?Cys?Asp?Pro?Asp?Lys?Tyr?Ser?Glu?Lys?Gly305?????????????????310?????????????????315?????????????????320Ser?Ser?Ser?Cys?Asn?Val?Arg?Pro?Ala?Cys?Thr?Asp?Lys?Asp?Tyr?Phe

325?????????????????330?????????????????335Tyr?Thr?His?Thr?Ala?Cys?Asp?Ala?Asn?Gly?Glu?Thr?Gln?Leu?Met?Tyr

340?????????????????345?????????????????350Lys?Trp?Ala?Lys?Pro?Lys?Ile?Cys?Ser?Glu?Asp?Leu?Glu?Gly?Ala?Val

355?????????????????360?????????????????365Lys?Leu?Pro?Ala?Ser?Gly?Val?Lys?Thr?His?Cys?Pro?Pro?Cys?Asn?Pro

370?????????????????375?????????????????380Gly?Phe?Phe?Lys?Thr?Asn?Asn?Ser?Thr?Cys?Gln?Pro?Cys?Pro?Tyr?Gly385?????????????????390?????????????????395?????????????????400Ser?Tyr?Ser?Asn?Gly?Ser?Asp?Cys?Thr?Arg?Cys?Pro?Ala?Gly?Thr?Glu

405?????????????????410?????????????????415Pro?Ala?Val?Gly?Phe?Glu?Tyr?Lys?Trp?Trp?Asn?Thr?Leu?Pro?Thr?Asn

420?????????????????425?????????????????430Met?Glu?Thr?Thr?Val?Leu?Ser?Gly?Ile?Asn?Phe?Glu?Tyr?Lys?Gly?Met

435?????????????????440?????????????????445Thr?Gly?Trp?Glu?Val?Ala?Gly?Asp?His?Ile?Tyr?Thr?Ala?Ala?Gly?Ala

450?????????????????455?????????????????460Ser?Asp?Asn?Asp?Phe?Met?Ile?Leu?Thr?Leu?Val?Val?Pro?Gly?Phe?Arg465?????????????????470?????????????????475?????????????????480Pro?Pro?Gln?Ser?Val?Met?Ala?Asp?Thr?Glu?Asn?Lys?Glu?Val?Ala?Arg

485?????????????????490?????????????????495Ile?Thr?Phe?Val?Phe?Glu?Thr?Leu?Cys?Ser?Val?Asn?Cys?Glu?Leu?Tyr

500?????????????????505?????????????????510Phe?Met?Val?Gly?Val?Asn?Ser?Arg?Thr?Asn?Thr?Pro?Val?Glu?Thr?Trp

515?????????????????520?????????????????525Lys?Gly?Ser?Lys?Gly?Lys?Gln?Ser?Tyr?Thr?Tyr?Ile?Ile?Glu?Glu?Asn

530?????????????????535?????????????????540Thr?Thr?Thr?Ser?Phe?Thr?Trp?Ala?Phe?Gln?Arg?Thr?Thr?Phe?His?Glu545?????????????????550?????????????????555?????????????????560Ala?Ser?Arg?Lys?Tyr?Thr?Asn?Asp?Val?Ala?Lys?Ile?Tyr?Ser?Ile?Asn

565?????????????????570?????????????????575Val?Thr?Asn?Val?Met?Asn?Gly?Val?Ala?Ser?Tyr?Cys?Arg?Pro?Cys?Ala

580?????????????????585?????????????????590Leu?Glu?Ala?Ser?Asp?Val?Gly?Ser?Ser?Cys?Thr?Ser?Cys?Pro?Ala?Gly

595?????????????????600?????????????????605Tyr?Tyr?Ile?Asp?Arg?Asp?Ser?Gly?Thr?Cys?His?Ser?Cys?Pro?Pro?Asn

610?????????????????615?????????????????620Thr?Ile?Leu?Lys?Ala?His?Gln?Pro?Tyr?Gly?Val?Gln?Ala?Cys?Val?Pro625?????????????????630?????????????????635?????????????????640Cys?Gly?Pro?Gly?Thr?Lys?Asn?Asn?Lys?Ile?His?Ser?Leu?Cys?Tyr?Asn

645?????????????????650?????????????????655Asp?Cys?Thr?Phe?Ser?Arg?Asn?Thr?Pro?Thr?Arg?Thr?Phe?Asn?Tyr?Asn

660?????????????????665?????????????????670Phe?Ser?Ala?Leu?Ala?Asn?Thr?Val?Thr?Leu?Ala?Gly?Gly?Pro?Ser?Phe

675?????????????????680?????????????????685Thr?Ser?Lys?Gly?Leu?Lys?Tyr?Phe?His?His?Phe?Thr?Leu?Ser?Leu?Cys

690?????????????????695?????????????????700Gly?Asn?Gln?Gly?Arg?Lys?Met?Ser?Val?Cys?Thr?Asp?Asn?Val?Thr?Asp705?????????????????710?????????????????715?????????????????720Leu?Arg?Ile?Pro?Glu?Gly?Glu?Ser?Gly?Phe?Ser?Lys?Ser?Ile?Thr?Ala

725?????????????????730?????????????????735Tyr?Val?Cys?Gln?Ala?Val?Ile?Ile?Pro?Pro?Glu?Val?Thr?Gly?Tyr?Lys

740?????????????????745?????????????????750Ala?Gly?Val?Ser?Ser?Gln?Pro?Val?Ser?Leu?Ala?Asp?Arg?Leu?Ile?Gly

755?????????????????760?????????????????765Val?Thr?Thr?Asp?Met?Thr?Leu?Asp?Gly?Ile?Thr?Ser?Pro?Ala?Glu?Leu

770?????????????????775?????????????????780Phe?His?Leu?Glu?Ser?Leu?Gly?Ile?Pro?Asp?Val?Ile?Phe?Phe?Tyr?Arg785?????????????????790?????????????????795?????????????????800Ser?Asn?Asp?Val?Thr?Gln?Ser?Cys?Ser?Ser?Gly?Arg?Ser?Thr?Thr?Ile

805?????????????????810?????????????????815Arg?Val?Arg?Cys?Ser?Pro?Gln?Lys?Thr?Val?Pro?Gly?Ser?Leu?Leu?Leu

820?????????????????825?????????????????830Pro?Gly?Thr?Cys?Ser?Asp?Gly?Thr?Cys?Asp?Gly?Cys?Asn?Phe?His?Phe

835?????????????????840?????????????????845Leu?Trp?Glu?Ser?Ala?Ala?Ala?Cys?Pro?Leu?Cys?Ser?Val?Ala?Asp?Tyr

850?????????????????855?????????????????860His?Ala?Ile?Val?Ser?Ser?Cys?Val?Ala?Gly?Ile?Gln?Lys?Thr?Thr?Tyr865?????????????????870?????????????????875?????????????????880Val?Trp?Arg?Glu?Pro?Lys?Leu?Cys?Ser?Gly?Gly?Ile?Ser?Leu?Pro?Glu

885?????????????????890?????????????????895Gln?Arg?Val?Thr?Ile?Cys?Lys?Thr?Ile?Asp?Phe?Trp?Leu?Lys?Val?Gly

900?????????????????905?????????????????910Ile?Ser?Ala?Gly?Thr?Cys?Thr?Ala?Ile?Leu?Leu?Thr?Val?Leu?Thr?Cys

915?????????????????920?????????????????925Tyr?Phe?Trp?Lys?Lys?Asn?Gln?Lys?Leu?Glu?Tyr?Lys?Tyr?Ser?Lys?Leu

930?????????????????935?????????????????940Val?Met?Asn?Ala?Thr?Leu?Lys?Asp?Cys?Asp?Leu?Pro?Ala?Ala?Asp?Ser945?????????????????950?????????????????955?????????????????960Cys?Ala?Ile?Met?Glu?Gly?Glu?Asp?Val?Glu?Asp?Asp?Leu?Ile?Phe?Thr

965?????????????????970?????????????????975Ser?Lys?Lys?Ser?Leu?Phe?Gly?Lys?Ile?Lys??Ser?Phe?Thr?Ser?Lys?Arg

980?????????????????985??????????????????990Thr?Pro?Asp?Gly?Phe?Asp?Ser?Val?Pro?Leu?Lys?Thr?Ser?Ser?Gly?Gly

995?????????????????1000????????????????1005Pro?Asp?Met?Asp?Leu

1010

<210>3

<211>677

<212>DNA

＜213〉people

<400>?3ttttttaatt?tacaaacaaa?gtgtgggtat?atttggcagg?tttgaggcaa?gcagaaagga????60gggtttgggt?actctataaa?aaaagatctg?aaattcaaac?atctgataag?gccacaatga???120agagatttcc?agcagtgggt?gttgcaggat?gctggcaccc?aaatcgccgc?acgttgcaaa???180ggtgctatgc?aaggtgagga?ggcaggtgag?gcaggcagtg?cctctcacag?gtccatgtct???240gggcctcctg?aggatgtctt?cagcggcact?gagtcaaatc?catcaggagt?cctcttggag???300gtaaatgatt?tgatcttccc?aaagagtgac?ttcttgctgg?taaagatgag?gtcgtcctct???360acatcctcgc?cttccatgat?ggcgcagtgt?cagctgctgg?caggtcacag?tccttgagag???420tagcattcat?caccagcttg?gagtacttgt?actctagttt?ttgattcttt?ttccaaaagt???480agcaggtcaa?gacggtgagc?aggatggcag?tacaggtgcc?tgcagagatg?cccactttca???540gccagaaatc?tatggttttg?cagatggtga?ctctctgctc?aggcagagaa?atgccaccag???600agcatagctt?gggttctcgc?cacacgtaag?tagtcttctg?gatcccagcc?acacagctgc???660tgacgatagc?atggtag??????????????????????????????????????????????????677

<210>4

<211>105

<212>PRT

＜213〉people

<400>4Tyr?His?Ala?Ile?Val?Ser?Ser?Cys?Val?Ala?Gly?Ile?Gln?Lys?Thr?Thr1????????????????5??????????????????10??????????????????15Tyr?Val?Trp?Arg?Glu?Pro?Lys?Leu?Cys?Ser?Gly?Gly?Ile?Ser?Leu?Pro

20??????????????????25??????????????????30Glu?Gln?Arg?Val?Thr?Ile?Cys?Lys?Thr?Ile?Asp?Phe?Trp?Leu?Lys?Val

35??????????????????40??????????????????45Gly?Ile?Ser?Ala?Gly?Thr?Cys?Thr?Ala?Ile?Leu?Leu?Thr?Val?Leu?Thr

50??????????????????55??????????????????60Cys?Tyr?Phe?Trp?Lys?Lys?Asn?Gln?Lys?Leu?Glu?Tyr?Lys?Tyr?Ser?Lys65??????????????????70??????????????????75??????????????????80Leu?Val?Met?Asn?Ala?Thr?Leu?Lys?Asp?Cys?Asp?Leu?Pro?Ala?Ala?Asp

85??????????????????90??????????????????95Thr?Ala?Pro?Ser?Trp?Lys?Ala?Arg?Met

100?????????????????105

<210>5

<211>9

<212>PRT

＜213〉artificial sequence

<400>5Arg?Leu?Trp?Arg?Leu?Leu?Leu?Trp?Ala?1???????????????5

<210>6

<211>9

<212>PRT

＜213〉artificial sequence

<400>6Ser?Leu?Ala?Asp?Arg?Leu?Ile?Gly?Val?1???????????????5

<210>7

<211>9

<212>PRT

＜213〉artificial sequence

<400>7Ser?Leu?Pro?Glu?Gln?Arg?Val?Thr?Ile?1???????????????5

<210>8

<211>9

<212>PRT

＜213〉artificial sequence

<400>8Lys?Leu?Cys?Ser?Gly?Gly?Ile?Ser?Leu?1???????????????5

<210>9

<211>9

<212>PRT

＜213〉artificial sequence

<400>9Ala?Ala?Ala?Cys?Pro?Leu?Cys?Ser?Val?1???????????????5

<210>10

<211>9

<212>PRT

＜213〉artificial sequence

<400>10Ser?Ala?Leu?Ala?Asn?Thr?Val?Thr?Leu?1???????????????5

<210>11

<211>9

<212>PRT

＜213〉artificial sequence

<400>11Phe?Val?Phe?Glu?Thr?Leu?Cys?Ser?Val?1???????????????5

<210>12

<211>9

<212>PRT

＜213〉artificial sequence

<400>12Glu?Leu?Pro?His?Gly?Phe?Ala?Ser?Leu?1???????????????5

<210>13

<211>9

<212>PRT

＜213〉artificial sequence

<400>13Leu?Ile?Phe?Thr?Ser?Lys?Lys?Ser?Leu?1???????????????5

<210>14

<211>9

<212>PRT

＜213〉artificial sequence

<400>14Lys?Leu?Glu?Tyr?Lys?Tyr?Ser?Lys?Leu?1???????????????5

<210>15

<211>9

<212>PRT

＜213〉artificial sequence

<400>15Lys?Thr?Ile?Asp?Phe?Trp?Leu?Lys?Val?1???????????????5

<210>16

<211>9

<212>PRT

＜213〉artificial sequence

<400>16Ser?Val?Ala?Asp?Tyr?His?Ala?Ile?Val?1???????????????5

<210>17

<211>9

<212>PRT

＜213〉artificial sequence

<400>17Leu?Leu?Leu?Pro?Gly?Thr?Cys?Ser?Asp?1???????????????5

<210>18

<211>9

<212>PRT

＜213〉artificial sequence

<400>18Ala?Leu?Ala?Asn?Thr?Val?Thr?Leu?Ala?1???????????????5

<210>19

<211>9

<212>PRT

＜213〉artificial sequence

<400>19Thr?Leu?Cys?Ser?Val?Asn?Cys?Glu?Leu?1???????????????5

<210>20

<211>9

<212>PRT

＜213〉artificial sequence

<400>20Asn?Thr?Asp?Glu?Cys?Thr?Ala?Thr?Leu?1???????????????5

<210>21

<211>9

<212>PRT

＜213〉artificial sequence

<400>21Ser?Leu?Pro?Asp?Pro?Val?Lys?Gly?Thr?1???????????????5

<210>22

<211>9

<212>PRT

＜213〉artificial sequence

<400>22Ser?Leu?Phe?Gly?Lys?Ile?Lys?Ser?Phe?1???????????????5

<210>23

<211>9

<212>PRT

＜213〉artificial sequence

<400>23Cys?Thr?Ala?Ile?Leu?Leu?Thr?Val?Leu?1???????????????5

<210>24

<211>9

<212>PRT

＜213〉artificial sequence

<400>24Ile?Val?Ser?Ser?Cys?Val?Ala?Gly?Ile?1???????????????5

<210>25

<211>9

<212>PRT

＜213〉artificial sequence

<400>25Lys?Met?Ser?Val?Cys?Thr?Asp?Asn?Val?1???????????????5

<210>26

<211>9

<212>PRT

＜213〉artificial sequence

<400>26Val?Leu?Val?Arg?Asn?Ile?Ala?Ile?Thr?1???????????????5

<210>27

<211>9

<212>PRT

＜213〉artificial sequence

<400>27Glu?Leu?Asn?Arg?Gly?Asn?Asn?Val?Leu?1???????????????5

<210>28

<211>9

<212>PRT

＜213〉artificial sequence

<400>28Ala?Thr?Leu?Met?Tyr?Ala?Val?Asn?Leu?1???????????????5

<210>29

<211>9

<212>PRT

＜213〉artificial sequence

<400>29Val?Ala?Val?Pro?His?Thr?Pro?Gly?Leu?1???????????????5

<210>30

<211>9

<212>PRT

＜213〉artificial sequence

<400>30Arg?Leu?Trp?Arg?Leu?Leu?Leu?Trp?Ala?1???????????????5

<210>31

<211>9

<212>PRT

＜213〉artificial sequence

<400>31Ser?Ala?Gly?Thr?Cys?Thr?Ala?Ile?Leu?1???????????????5

<210>32

<211>9

<212>PRT

＜213〉artificial sequence

<400>32Gly?Ile?Ser?Leu?Pro?Glu?Gln?Arg?Val?1???????????????5

<210>33

<211>9

<212>PRT

＜213〉artificial sequence

<400>33Lys?Thr?Val?Pro?Gly?Ser?Leu?Leu?Leu?1???????????????5

<210>34

<211>9

<212>PRT

＜213〉artificial sequence

<400>34Arg?Leu?Ile?Gly?Val?Thr?Thr?Asp?Met?1???????????????5

<210>35

<211>9

<212>PRT

＜213〉artificial sequence

<400>35Thr?Leu?Ala?Gly?Gly?Pro?Ser?Phe?Thr?1???????????????5

<210>36

<211>9

<212>PRT

＜213〉artificial sequence

<400>36Tyr?Ile?Ile?Glu?Glu?Asn?Thr?Thr?Thr?1???????????????5

<210>37

<211>9

<212>PRT

＜213〉artificial sequence

<400>37Ile?Ala?Ile?Thr?Gly?Val?Ala?Tyr?Thr?1???????????????5

<210>38

<211>9

<212>PRT

＜213〉artificial sequence

<400>38Val?Thr?Gln?Gly?Thr?Gly?Pro?Glu?Leu?1???????????????5

<210>39

<211>10

<212>PRT

＜213〉artificial sequence

<400>39Ser?Leu?Phe?Gly?Lys?Ile?Lys?Ser?Phe?Thr?1???????????????5??????????????????10

<210>40

<211>10

<212>PRT

＜213〉artificial sequence

<400>40Ala?Ile?Val?Ser?Ser?Cys?Val?Ala?Gly?Ile?1???????????????5??????????????????10

<210>41

<211>10

<212>PRT

＜213〉artificial sequence

<400>41Ser?Ala?Ala?Ala?Cys?Pro?Leu?Cys?Ser?Val?1???????????????5??????????????????10

<210>42

<211>10

<212>PRT

＜213〉artificial sequence

<400>42His?Leu?Glu?Ser?Leu?Gly?Ile?Pro?Asp?Val?1???????????????5??????????????????10

<210>43

<211>10

<212>PRT

＜213〉artificial sequence

<400>43Lys?Ile?Tyr?Ser?Ile?Asn?Val?Thr?Asn?Val?1???????????????5??????????????????10

<210>44

<211>10

<212>PRT

＜213〉artificial sequence

<400>44Ser?Leu?Ala?Asp?Arg?Leu?Ile?Gly?Val?Thr?1???????????????5??????????????????10

<210>45

<211>10

<212>PRT

＜213〉artificial sequence

<400>45Ile?Leu?Lys?Ala?His?Gln?Pro?Tyr?Gly?Val?1???????????????5??????????????????10

<210>46

<211>10

<212>PRT

＜213〉artificial sequence

<400>46Val?Met?Ala?Asp?Thr?Glu?Asn?Lys?Glu?Val?1???????????????5??????????????????10

<210>47

<211>10

<212>PRT

＜213〉artificial sequence

<400>47Leu?Leu?Trp?Ala?Gly?Thr?Ala?Phe?Gln?Val?1???????????????5??????????????????10

<210>48

<211>10

<212>PRT

＜213〉artificial sequence

<400>48Gly?Thr?Cys?Thr?Ala?Ile?Leu?Leu?Thr?Val?1???????????????5??????????????????10

<210>49

<211>10

<212>PRT

＜213〉artificial sequence

<400>49Ile?Thr?Ser?Pro?Ala?Glu?Leu?Phe?His?Leu?1???????????????5??????????????????10

<210>50

<211>10

<212>PRT

＜213〉artificial sequence

<400>50Leu?Ile?Gly?Val?Thr?Thr?Asp?Met?Thr?Leu?1???????????????5??????????????????10

<210>51

<211>10

<212>PRT

＜213〉artificial sequence

<400>51Thr?Leu?Pro?Thr?Asn?Met?Glu?Thr?Thr?Val?1???????????????5??????????????????10

<210>52

<211>10

<212>PRT

＜213〉artificial sequence

<400>52Leu?Met?Tyr?Lys?Trp?Ala?Lys?Pro?Lys?Ile?1???????????????5??????????????????10

<210>53

<211>10

<212>PRT

＜213〉artificial sequence

<400>53Asp?Leu?Ile?Phe?Thr?Ser?Lys?Lys?Ser?Leu?1???????????????5??????????????????10

<210>54

<211>10

<212>PRT

＜213〉artificial sequence

<400>54Gly?Leu?Lys?Tyr?Phe?His?His?Phe?Thr?Leu?1???????????????5??????????????????10

<210>55

<211>10

<212>PRT

＜213〉artificial sequence

<400>55Gly?Thr?Lys?Asn?Asn?Lys?Ile?His?Ser?Leu?1???????????????5??????????????????10

<210>56

<211>10

<212>PRT

＜213〉artificial sequence

<400>56Ser?Asp?Asn?Asp?Phe?Met?Ile?Leu?Thr?Leu?1???????????????5??????????????????10

<210>57

<211>10

<212>PRT

＜213〉artificial sequence

<400>57Leu?Val?Arg?Asn?Ile?Ala?Ile?Thr?Gly?Val?1???????????????5??????????????????10

<210>58

<211>10

<212>PRT

＜213〉artificial sequence

<400>58Gly?Leu?Cys?Thr?Ser?Leu?Pro?Asp?Pro?Val?1???????????????5??????????????????10

<210>59

<211>10

<212>PRT

＜213〉artificial sequence

<400>59Thr?Leu?Lys?Asp?Cys?Asp?Leu?Pro?Ala?Ala?1???????????????5??????????????????10

<210>60

<211>10

<212>PRT

＜213〉artificial sequence

<400>60Val?Ile?Phe?Phe?Tyr?Arg?Ser?Asn?Asp?Val?1???????????????5??????????????????10

<210>61

<211>10

<212>PRT

＜213〉artificial sequence

<400>61Ser?Ile?Thr?Ala?Tyr?Val?Cys?Gln?Ala?Val?1???????????????5??????????????????10

<210>62

<211>10

<212>PRT

＜213〉artificial sequence

<400>62Ser?Val?Cys?Thr?Asp?Asn?Val?Thr?Asp?Leu?1???????????????5??????????????????10

<210>63

<211>10

<212>PRT

＜213〉artificial sequence

<400>63Ser?Leu?Cys?Gly?Asn?Gln?Gly?Arg?Lys?Met?1???????????????5??????????????????10

<210>64

<211>10

<212>PRT

＜213〉artificial sequence

<400>64Asn?Met?Glu?Thr?Thr?Val?Leu?Ser?Gly?Ile?1???????????????5??????????????????10

<210>65

<211>10

<212>PRT

＜213〉artificial sequence

<400>65Asn?Ile?Ala?Ile?Thr?Gly?Val?Ala?Tyr?Thr?1???????????????5??????????????????10

<210>66

<211>10

<212>PRT

＜213〉artificial sequence

<400>66Gly?Ile?Arg?Phe?Asp?Glu?Trp?Asp?Glu?Leu?1???????????????5??????????????????10

<210>67

<211>10

<212>PRT

＜213〉artificial sequence

<400>67Phe?His?Phe?Leu?Trp?Glu?Ser?Ala?Ala?Ala?1???????????????5??????????????????10

<210>68

<211>9

<212>PRT

＜213〉artificial sequence

<400>68Phe?Leu?Trp?Glu?Ser?Ala?Ala?Ala?Cys?1???????????????5

Claims (37)

1. the isolated polypeptide that contains following aminoacid sequence, the full length sequence of the aminoacid sequence of this aminoacid sequence and SEQ IDNO:2 has at least 70% identity.

2. the isolated polypeptide of claim 1, wherein the aminoacid sequence of aminoacid sequence and SEQ ID NO:2 has at least 95% identity.

3. the isolated polypeptide of claim 1 contains the aminoacid sequence of SEQ ID NO:2.

4.SEQ the isolated polypeptide of ID NO:2.

5. contain each the polypeptide of immunogenic fragments of polypeptide of claim 1-4, the immunogen activity of the wherein said immunogenic fragments polypeptide with SEQ ID NO:2 basically is identical.

6. each polypeptide of claim 1-5, wherein said polypeptide are the parts of larger fusion protein more.

7. each polypeptide of claim 1-6 stops with the carrier proteins chemistry and to close.

8. each separation polynucleotide of polypeptide of coding claim 1-6.

9. contain the separating polynucleotide or separate polynucleotide complementary nucleotide sequence with this of nucleotide sequence that coding and the full length sequence of the aminoacid sequence of SEQ ID NO:2 have the polypeptide of at least 70% identity.

10. contain the separating polynucleotide or separate polynucleotide complementary nucleotide sequence with this of nucleotide sequence that has at least 70% identity with whole coding regions of the nucleotide sequence of the polypeptide of coding SEQ ID NO:2.

11. contain the separating polynucleotide or separate polynucleotide complementary nucleotide sequence with this of nucleotide sequence that has the polypeptide of at least 70% identity with the full length sequence of the nucleotide sequence of SEQ ID NO:1.

12. the separation polynucleotide of each qualification of claim 8-11, wherein identity is at least 95%.

13. isolating polynucleotide are selected from:

(a) contain the polynucleotide of nucleotide sequence of the polypeptide of coding SEQ ID NO:2;

(b) polynucleotide of SEQ ID NO:1; With

(c) polynucleotide by under tight hybridization conditions, obtaining with the suitable library of label probe screening, described probe has sequence or its fragment of SEQ ID NO:1, the albumen that described polynucleotide encoding albumen a kind of and SEQ ID NO:2 has similar immunogen characteristic

Or with described polynucleotide complementary nucleotide sequence.

14. contain claim 8-13 each the separation polynucleotide expression vector or the reorganization live microorganism.

15. contain each the host cell of separation polynucleotide of the expression vector of claim 14 or claim 8-13.

16. the method for the polypeptide of production claim 1-7 is included in the host cell of cultivating claim 15 under the condition that is enough to produce described polypeptide and by reclaiming this polypeptide in the substratum.

17. contain significant quantity claim 1-7 each polypeptide and the vaccine of pharmaceutically acceptable carrier.

18. contain significant quantity claim 8-13 each polynucleotide and the vaccine of pharmaceutically acceptable carrier.

19. contain the antigen presenting cell of significant quantity and the vaccine of pharmaceutically acceptable carrier, described cell by external load claim 1-7 each polypeptide and modified or external by genetic modification to express the polypeptide of claim 1-7.

20. each vaccine of claim 17-19 also comprises the TH-1 inducing adjuvant.

21. the vaccine of claim 20, wherein the TH-1 inducing adjuvant is selected from: the mixture of 3D-MPL, QS21, QS21 and cholesterol and CpG oligonucleotide.

22. to claim 1-5 each polypeptide or the antibody of immune fragment immunologic opsonin.

23. identify to stimulate or suppress each the screening method of compound of polypeptide function of claim 1-5, comprise the following method that is selected from:

(a) by direct or indirect and candidate compound bonded mark, the mensuration candidate compound combines with described polypeptide (or the cell of this polypeptide of load or film) or its fusion rotein;

(b) when mark competition thing exists, the mensuration candidate compound combines with described polypeptide (or the cell of this polypeptide of load or film) or its fusion rotein;

(c) use is measured the signal whether candidate compound causes this polypeptide to activate or suppress to produce to the cell or the suitable detection system of cytolemma of this polypeptide of load;

(d) with candidate compound with contain each the solution of polypeptide of claim 1-7 and mix and form mixture, measure the polypeptide active in the mixture, relatively the activity of mixture and standard substance; Or

(e) use as the mRNA of coding said polypeptide in the ELISA measuring candidate compound pair cell and as described in the influence of generation of polypeptide.

24. method by immunoprophylaxis or treatment treatment experimenter, comprise with claim 1-7 each polypeptide or claim 8-13 each polynucleotide and the cells in vitro incubation of immune system, external evoked at each the immunne response of molecule of claim 1-5, then these activatory immunocytes are imported again in the Mammals with the treatment disease.

25. the method for claim 24, wherein treatment is at ovarian cancer or colorectal carcinoma.

26. the agonist of the polypeptide of claim 1-5 or antagonist.

27. a compound, it is:

(a) agonist of the polypeptide of claim 1-5 or antagonist;

(b) the separation polynucleotide of claim 8-13; Or

(c) regulate the nucleic acid molecule that each the nucleotide sequence of polypeptide of coding claim 1-5 is expressed;

It is used for the treatment of.

28. among the diagnosis experimenter with claim 1-5 each polypeptide active or express diseases associated or, comprise existence or its amount of analysis from polypeptide described in this experimenter's the sample to the method for the susceptibility of disease.

29. it is active or express diseases associated or, comprise existence or its amount of analysis among the diagnosis experimenter from polynucleotide described in this experimenter's the sample to the method for the susceptibility of disease with each polynucleotide of claim 8-13.

30. among the diagnosis experimenter with claim 1-5 each polypeptide active or express relevant colorectal carcinoma or, comprise existence or its amount of analysis from polypeptide described in this experimenter's the sample to the method for the susceptibility of colorectal carcinoma.

31. it is active or express diseases associated or, comprise existence or its amount of analysis among the diagnosis experimenter from polynucleotide described in this experimenter's the sample to the method for the susceptibility of disease with each polynucleotide of claim 8-13.

32. isolating polynucleotide are selected from:

(a) contain with the full length sequence of SEQ ID NO:3 have at least 70% identity nucleotide sequence separate polynucleotide;

(b) contain the separation polynucleotide of the polynucleotide of SEQ ID NO:3;

(c) polynucleotide of SEQ ID NO:3.

33. contain the expression vector of claim 14 or the living vaccine composition of reorganization live microorganism.

34. each the purposes of polynucleotide in the medicine of preparation treatment cancer of claim 8-13.

35. each the purposes of polynucleotide in the medicine of preparation treatment colorectal carcinoma of claim 8-13.

36. each the purposes of polypeptide in the medicine of preparation treatment cancer of claim 1-7.

37. each the purposes of polypeptide in the medicine of preparation treatment colorectal carcinoma of claim 1-7.

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