CN1840178B - Tumour-specific animal proteins - Google Patents

Abstract

CASB7439 polypeptides and polynucleotides, immunogenic compositions comprising them and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CASB7439 polypeptides and polynucleotides in diagnostics, and vaccines for prophylactic and therapeutic treatment of cancers, particularly colorectal cancers, autoimmune diseases, and related conditions.

Description

Tumour-specific animal proteins

The application is the divisional application of following application: the applying date: February 16 calendar year 2001; Application number: 01808411.7 (PCT/EP01/01779); Denomination of invention: " tumour-specific animal proteins ".

Invention field

The present invention relates to for inducing Pharmaceutical composition and the method for the immunne response of tumor associated antigen.More particularly, the present invention relates to polynucleotide (being called CASB7439 polynucleotide herein), by the polypeptide (being called CASB7439 polypeptide herein) of their codings, for the production of their recombined material and method.On the other hand, the present invention relates to the using method of such polypeptide and polynucleotide, comprise the therapy for the treatment of cancer, more especially colorectal carcinoma and autoimmune disease and other associated conditions.On the other hand, the Pharmaceutical composition that the present invention relates to contain CASB7439 polypeptide and polynucleotide, relate to the production method of this compositions and the application at field of medicaments thereof.Again on the one hand, the present invention relates to identify the method for agonist and antagonist/inhibitor and use the method for the disease that identified compounds for treating is uneven relevant to CASB7439 polypeptide with material provided by the invention.Another aspect, the present invention relates to for detection of with the diagnostic analysis of the disease of inappropriate CASB7439 polypeptide active or Horizontal correlation.

Background technology

Polypeptide of the present invention and polynucleotide are considered to the important immunogen of the preventative or therapeutic immunization of specificity for tumor, because compared with expression in normal cell, they are specific expressed or height overexpression in tumor, therefore can by antigen specific immune mechanism targeting they, cause destroying tumor cell.They can also be used for the existence of diagnosing tumour cell.In addition, they in some cases unsuitable expression can cause induction autoimmune, i.e. unsuitable immunne response, by with described polypeptide or polynucleotide suitably vaccination may correct described unsuitable immunne response.In this respect, for our object, most important biological activity is antigen active and the immunogenicity activity of polypeptide of the present invention.A peptide species of the present invention also may show at least one other biological activity of a kind of CASB7439 polypeptide, and it may be counted as and be different from the therapeutic of the therapeutic relevant to described immunne response or preventative interference or the target of preventative interference.

Summary of the invention

First aspect, the present invention relates to CASB7439 polypeptide.This peptide comprises the polypeptide of separation, one section of aminoacid sequence that described polypeptide comprises is at SEQ ID NO:2, SEQ ID NO:3, SEQID NO:7, SEQ ID NO:10, SEQ ID NO:11, in the total length of SEQ ID NO:12 or SEQ IDNO:14 respectively with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, the aminoacid sequence of SEQ ID NO:12 or SEQ ID NO:14 has at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity again, most preferably 97-99% homogeneity at least, prerequisite is that the polypeptide of described separation is not SEQ ID NO:2, SEQ ID NO:12 or SEQ IDNO:14.This peptide species comprises the amino acid whose polypeptide containing SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:11.

Other peptide of the present invention comprises the polypeptide of separation, wherein said aminoacid sequence is at SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, in the total length of SEQ ID NO:12 or SEQ ID NO:14 respectively with SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, the aminoacid sequence of SEQ ID NO:12 or SEQ ID NO:14 has at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity again, most preferably 97-99% homogeneity at least, prerequisite is that the polypeptide of described separation is not SEQ ID NO:2, SEQID NO:12 or SEQ ID NO:14.This peptide species comprises the polypeptide of SEQ ID NO:3, SEQ IDNO:7, SEQ ID NO:10 and SEQ ID NO:11.

Preferably aforementioned polypeptides produces by restructuring.Be most preferably purified according to polypeptide of the present invention, and do not basically contain the material of any other albumen or contaminative Hosts.

Other peptide of the present invention comprises by the polypeptide that is included in the separation of the polynucleotide encoding of contained sequence in SEQ ID NO:1.

The present invention also provides the immunogenic fragments of CASB7439 polypeptide, be a continuous part of CASB7439 polypeptide, described immunogenic fragments has and the same or analogous immunogenicity characteristic of the polypeptide that comprises following aminoacid sequence: SEQ ID NO:2, SEQ ID NO:3, SEQID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 or SEQ IDNO:14.That is to say, described fragment (if necessary, with carrier coupling or as compared with a part for larger fusion protein) can produce the immunne response of the described CASB7439 polypeptide of identification.Such immunogenic fragments can comprise the CASB7439 polypeptide that for example lacks N-end targeting sequencing, membrane spaning domain or C-end anchors and domain.One preferred aspect, the whole ectodomain substantially that comprises polypeptide according to CASB7439 immunogenic fragments of the present invention, described polypeptide is at SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, in the total length of SEQ ID NO:12 or SEQ ID NO:14 respectively with SEQID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, the aminoacid sequence of SEQ ID NO:12 or SEQ ID NO:14 has at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity again, most preferably 97-99% homogeneity at least.Preferably comprise at least one epi-position according to immunogenic fragments of the present invention.

The fragments of peptides that mixes CASB7439 epi-position conventionally comprises and derives from least 7 of SEQ ID NO:2, preferably 9 or 10 continuous amino acids.Preferred epi-position is shown in SEQ ID NO:16 to SEQ ID NO:33.

The peptide that mixes these epi-positions forms a preferred aspect of the present invention.The mimic epitope (mimotope) identical with the feature of these epi-positions and comprise these mimic epitopes, produce and epi-position in CASB7439 molecule environment has the immunogen of the immunne response of cross reaction, also forms a part of the present invention.

Therefore, the present invention includes the peptide of the separation that comprises these epi-positions itself and any mimic epitope thereof.The implication of mimic epitope be defined as to natural CASB7439 epi-position enough similar, so that can be identified the entity of the antibody recognition of described natural molecule; (Gheysen, H.M. etc., 1986, Synthetic peptides as antigens.Wiley; Chichester, Ciba foundationsymposium 119, the 130-149 pages; Gheysen, H.M., 1986, MolecularImmunology, 23,7,709-715); Or when with suitable carrier coupling, can produce the entity of antibody, wherein said antibody and described natural molecule have cross reaction.

The peptide mimic epitope of above identified epi-position can, by adding, lack or replacing selected aminoacid, be designed to for specific purpose.Thereby, can modify peptide of the present invention, put together with protein carrier being convenient to.For example, for some chemically conjugated method, may it is desirable to described epi-position and comprise a terminal cysteine.In addition, for the peptide of puting together with protein carrier, may it is desirable to comprise a hydrophobicity end of puting together end away from described peptide, so that the free unconjugated end of described peptide retains and the surface association of described carrier protein.This has reduced the conformational freedom of described peptide, thus increased with the peptide conformation existing the described complete molecule in the situation that probability that the most similarly conformation is presented described peptide.For example, described peptide can be changed to and have a N-end cysteine and a C-end hydrophobic amide tail.On the other hand, can carry out interpolation or the replacement of one or more described amino acid whose D-stereoisomer forms, to produce useful derivant, for example, strengthen the stability of described peptide.Those of skill in the art will recognize that this modified peptide or mimic epitope can be all or part of non-peptide mimic epitopes, wherein said composition residue is not necessarily limited to 20 kinds of naturally occurring aminoacid.In addition, can be by technology known in the art by its cyclisation, described peptide is limited in to the conformation of the shape of similar described peptide sequence in complete molecule environment closely.A method for optimizing of peptide cyclisation is comprised and adds a pair of cysteine residues, to allow to form a disulphide bridges.

In addition, those of skill in the art will recognize that mimic epitope of the present invention or immunogen can be larger than the epi-position of above-mentioned evaluation, therefore can comprise sequence disclosed herein.Therefore, mimic epitope of the present invention can be included in an end or two ends add many other natural residue N and/C holds extension.Described peptide mimic epitope can be also the antitone sequence (retrosequence) of native sequences, because described sequence direction is contrary; Or described sequence can all or at least partly be made up of D-stereoisomer aminoacid (inversion sequence (inverso sequence)).In addition, described peptide sequence can be (retro-inverso) of trans-inversion in feature, because described sequence direction is contrary, and described aminoacid is D-stereoisomer form.The advantage of trans-inversion peptide is like this nonself, therefore can overcome the problem of the self-tolerance in immune system.

On the other hand, adopt the multiple technologies such as display technique of bacteriophage (EP 0 552 267 B1), use the antibody that himself can be combined with epi-position of the present invention can identify peptide mimic epitope.This technology produces the peptide sequence of a large amount of simulation native peptides structures, therefore can with anti-native peptides antibodies, but not necessarily self and described native peptides are shared significant sequence homology.The method may be very favorable, because make the peptide of likely identifying that immunogenicity characteristic strengthens, or can overcome any potential autoantigen Commpensation And Adaptation that may be relevant to the described native sequence polypeptide of application, in addition, in view of it has shared chemical characteristic in institute's identification polypeptide mimic epitope sequence, this technology makes it possible to identify the recognition mode of various native peptides.

Can make described peptide and immunogenic carrier carry out covalent coupling by method well-known in the art.Therefore, for example, for direct covalent coupling, likely utilize carbodiimide, glutaraldehyde or (N-[γ-dimaleoyl imino butyryl acyloxy] succinimide ester, utilize common commercially available for example CDAP of Heterobifunctional joint and SPDP (adopting manufacturer's explanation)).After coupling reaction, described immunogen can easily separate and purification by dialysis, gel filtration, fractionated method etc.

In immunogen of the present invention, bearer type used is that those skilled in the art easily know.It is auxiliary that the function of described carrier is to provide cytokine, to help the immunne response of induction for described peptide.The non-property the exhausted catalog that can be used for carrier of the present invention comprises: keyhole the purfied protein derivative (PPD) of hemocyanin (KLH), for example bovine serum albumin of serum albumin (BSA), deactivation bacteriotoxin (for example tetanus toxin or diphtheria toxin, diphtherotoxin (TT and DT)) or its recombinant fragment (for example, the easy bit architecture structure territory of the domain 1 of the fragment C of TT or DT) or tuberculin.On the other hand, described mimic epitope or epi-position can be directly and liposome vectors put together, the immunogen that can provide T-cell auxiliary is provided described liposome vectors in addition.Mimic epitope is preferably about 1: 1 to 20: 1 with the ratio of carrier, and each carrier preferably should carry 3-15 peptide.

In one embodiment of the invention, a kind of preferred carrier is the D albumen (EP 0 594 610 B1) of Haemophilus influenzae (Haemophilus influenzae).D albumen be a kind of IgD that derives from Haemophilus influenzae in conjunction with albumen, and the patented power of Forsgren (WO 91/18926, Granted publication number (granted) EP 0 594 610 B1).In some cases, for example, in the former expression system of recombinant immune, may preferably utilize the fragment of D albumen, for example 1/3rd of D albumen (comprising 100-110 aminoacid of D albumen N-end (GB 9717953.5)).

Another preferred method of presenting peptide of the present invention is to present in the environment of recombinant fusion molecule.For example, EP 0 421 635 B have described using chimeric hepadnavirus core antigen particles and have presented the exogenous peptide sequence in virus-like particle.Therefore, immunogen of the present invention can be included in the peptide of presenting in the chimeric granule being made up of hepatitis B core antigen.In addition, described recombination fusion protein can comprise mimic epitope of the present invention and a kind of carrier protein, the NS1 of for example influenza virus.For any recombinant expressed albumen that forms part of the present invention, the described immunogenic nucleic acid of encoding also forms one aspect of the present invention.

Peptide used in the present invention can be easily synthetic by solid phase method well-known in the art.Suitable synthetic can being undertaken by utilization " T-boc " or " F-moc " method.Cyclic peptide can utilize the polyamide in well-known " F-moc " method and full-automatic device to synthesize by solid phase method.Or, one skilled in the art will recognize that and manually carry out the essential laboratory method of described method.The technology of solid phase synthesis and method be described in E.Atherton and R.C.Sheppard's " Solid Phase Peptide Synthesis:A Practical Approach ", the IRL of OxfordUniversity Press publishes (1989).Or described peptide can produce by recombination method, be included in the nucleic acid molecules of expressing the described mimic epitope of coding in antibacterial or mammal cell line, the then expressed mimic epitope of purification.The recombination and expression techniques of peptide and albumen is known in the art, and is described in Maniatis, T., Fritsch, E.F. and Sambrook etc., Molecular cloning, a laboratory manual, the second edition; Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York (1989).

The preparation method of polypeptide described herein is provided in an embodiment more of the present invention.Method of the present invention can be undertaken by conventional recombinant technique, and described conventional recombinant technique is for example described in Maniatis etc., Molecular Cloning-A Laboratory Manual; In Cold SpringHarbor (1982-1989).Therefore, provide preparation according to the method for polypeptide of the present invention, described method is included under the condition that is enough to produce described polypeptide and cultivates host cell, reclaims described polypeptide from described culture medium.Specifically, method of the present invention may preferably include the following step:

I) preparation can be in host cell replication form or the integrated expression vector of expressible dna polymer, the nucleotide sequence that described DNA polymer comprises encoding said proteins or its immunogenic derivatives;

Ii) with described carrier transformed host cell;

Iii) allowing to express the host cell of cultivating described conversion under the condition of described DNA polymer, to produce described albumen; With

Iv) reclaim described albumen.

Polypeptide of the present invention or immunogenic fragments can be " maturation " albumen forms, or can be that larger albumen is as a part for precursor protein or fusion rotein.Comprise that the additional amino acid sequence that comprises following sequence is normally favourable: secretion sequence or targeting sequencing, former sequence (pro-sequences), contribute to the sequence of purification to be conducive to other sequence of stability as polyhistidine residue or in recombinant production process.In addition, also consider to add allogenic polypeptide or lipid tail or polynucleotide sequence, to increase the immunogenic potential of final molecule.

On the one hand, the present invention relates to genetic engineering soluble fusion protein, described fusion rotein comprises polypeptide of the present invention or its fragment, and the different piece of the constant region of the immunoglobulin of different subclass (IgG, IgM, IgA, IgE) heavy chain or light chain.As immunoglobulin, preferably the constant portion of the heavy chain of IgG (particularly IgG1), wherein merges in hinge region.In a particular, can just can remove Fc part by the cutting sequence that adds available factor Xa excision.In addition, the present invention relates to prepare by genetic engineering the method for these fusion rotein, and the application of these fusion rotein in drug screening, diagnosis and treatment.A particularly preferred aspect of the present invention relates to application polypeptide or the treatment of polynucleotide production immunotherapeutical suffers from or the patient's of easy cancer stricken, especially colon cancer or other colon dependency tumor or disease vaccine.The polynucleotide that also relate on the one hand the more such fusion rotein of coding of the present invention.In international patent application no WO94/29458 and WO94/22914, can find the example of fusion protein technology.

Described albumen can be chemically conjugated or as fusion protein expression, to make compared with the albumen not merging, the level producing in expression system improves.Described fusion partner can assist to provide t helper cell epi-position (Immune Fusion gametophyte), the t helper cell epi-position identified by the mankind preferably, or described fusion partner contributes to express described albumen (expression enhancer) than natural recombiant protein higher yield.Best described fusion partner is Immune Fusion gametophyte, is again to express to strengthen gametophyte.

Fusion partner comprises the D albumen of Haemophilus influenzae B and the non-structural protein NS 1 (hemagglutinin) of influenza virus.Another kind of Immune Fusion gametophyte is the albumen that is called LYTA.Preferably use the C-end portion of described molecule.Lyta derives from streptococcus pneumoniae (Streptococcuspneumoniae), it synthesizes N-acetyl-ALANINE amidase-amidase LYTA (by lytA gene code { Gene, 43 (1986) 265-272 pages }), i.e. the autolysin of some key in specificity degraded Peptidoglycan skeleton.Be responsible for choline or some cholinomimetics as the affinity of DEAE in the C-end structure territory of LYTA albumen.Utilize this feature development to can be used for the escherichia coli C-LYTA expression plasmid of expressed fusion protein.The purification { Biotechnology:10, (1992) 795-798 pages } of the hybrid albumen that comprises C-LYTA fragment at its aminoterminal has been described.Can utilize the repetitive sequence part that originates in residue 178 that is present in Lyta molecule C-end, for example residue 188-305.

The present invention also comprises the xenogenesis form (also referred to as straight homologues form) of aforementioned polypeptide, described xenogenesis form refers to mankind's antigen (also referred to as autoantigen) to have the roughly antigen of sequence homogeneity, and described antigen is as the reference antigen that derives from different non-human species.Aspect this, described roughly homogeneity refer to when sequence this area as numerous sequence alignment albumen in any in the concordance of an aminoacid sequence and another aminoacid sequence or a polynucleotide sequence and another polynucleotide sequence while arranging with optimal sequence comparison.So-called roughly homogeneity refers to that the sequence homogeneity between comparative sequences is at least 70-95%, preferably at least 85-95%, 90-95% at least most preferably.Therefore, according to the present invention, xenogenesis CASB7439 polypeptide will be the CASB7439 polypeptide that is xenogenesis for people CASB7439, and in other words, xenogenesis CASB7439 polypeptide is separated from inhuman species.In a preferred embodiment, described polypeptide separates from mice, rat, pig or Rhesus Macacus, most preferably separates from mice or rat.Therefore, the present invention also provides and in human body, induces the method for the immunne response of people CASB7439, described method comprises the compositions of the xenogenesis form of described people CASB7439 as described herein that comprises that gives described curee's effective dose, and the aminoacid sequence of described people CASB7439 is as shown in the arbitrary sequence in sequence SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQID NO:10 or SEQ ID NO:11.A preferred embodiment is to use the xenogenesis CASB7439 separating from mice, rat, pig or Rhesus Macacus to induce the method for the immunne response of people CASB7439.To use the antigen composition that comprises the live virus expression system of expressing described heteroantigen according to the another kind of method for optimizing of induce immune response of the present invention.The sequence of preferred xenogenesis CASB7439 polypeptide is shown in SEQ ID NO:12 (mice) or SEQ ID NO:14 (rat).

The xenogenesis CASB7439 polypeptide separating is conventionally shared roughly sequence similarity, comprises in the total length that is included in SEQ ID NO:12 or SEQ ID NO:14 with the aminoacid sequence of SEQ ID NO:12 or SEQ ID NO:14 and has at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity, the isolated polypeptide of the aminoacid sequence of 97-99% homogeneity at least most preferably again.Therefore, the immunogenic fragments of the polypeptide that described xenogenesis polypeptide comprises SEQ IDNO:12 or SEQ ID NO:14, the immunogenicity activity of wherein said immunogenic fragments is substantially identical with the polypeptide of SEQ ID NO:12 or SEQ ID NO:14.In addition, described xenogenesis CASB7439 polypeptide can be such fragment: be selected from the aminoacid sequence shown in SEQ IDNO:12 or SEQ ID NO:14 at least about 20 continuous amino acids, preferably approximately 30, more preferably from about 50, more preferably from about 100,150 continuous amino acids most preferably from about again.More particularly, xenogenesis CASB7439 fragment can retain more macromolecular some functional characteristic shown in SEQ ID NO:12 or SEQ ID NO:14, immunocompetence preferably, and can be used in method as herein described (for example can be used for Pharmaceutical composition or vaccine combination, diagnostics is medium).Specifically, described fragment can cause the immunne response for mankind's homologue, for example, produce the cross reacting antibody reacting with self mankind CASB7439 form shown in arbitrary SEQ ID NO:2.In a specific embodiment, xenogenesis polypeptide of the present invention can be a part for larger fusions, and described fusions comprises xenogenesis CASB7439 polypeptide or its fragment and heterologous protein or protein part as fusion partner as above.

The present invention also comprises the variant of aforementioned polypeptide, thus by conserved amino acid replace a residue by another have similar characteristic residue replace and the polypeptide different from reference substance.Conventionally such replacement occurs between Ala, Val, Leu and Ile; Between Ser and Thr; Between acidic residues Asp and Glu; Between Asn and Gln; And between alkaline residue Lys and Arg; Or between aromatic residue Phe and Tyr.Particularly preferably wherein several, 5-10,1-5,1-3,1-2 or 1 variant that aminoacid replaces, lacks or add with any combination.

Can prepare in any suitable manner polypeptide of the present invention.Such polypeptide comprises the polypeptide of the naturally occurring polypeptide of separation, the polypeptide that restructuring produces, synthetic generation or passes through the polypeptide of the combination results of these methods.Well-known in the art for the preparation of the method for such polypeptide.

On the other hand, the present invention relates to CASB7439 polynucleotide.Such polynucleotide comprise the polynucleotide containing the separation of the nucleotide sequence of coded polypeptide, and described polypeptide has at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity again with the aminoacid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ IDNO:10 or SEQ ID NO:11 respectively in the total length of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:11.At this on the one hand, highly preferably have the coded polypeptide of at least 97% homogeneity, and more highly preferably have at least coded polypeptide of 98-99% homogeneity, topnotch preferably has the coded polypeptide of at least 99% homogeneity.

Other polynucleotide of the present invention comprise the polynucleotide of separation, and one section of nucleotides sequence that described polynucleotide comprise is listed on complete coding region with the nucleotide sequence of the polypeptide of coding SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:11 has at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity again.At this on the one hand, highly preferably have the polynucleotide of at least 97% homogeneity, and more highly preferably have at least polynucleotide of 98-99% homogeneity, topnotch preferably has the polynucleotide of at least 99% homogeneity.

Other polynucleotide of the present invention comprise the polynucleotide of separation, one section of nucleotides sequence that described polynucleotide comprise is listed in the total length of described sequence and SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:9 or at SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, in the total length of the described coded sequence of SEQ ID NO:8 or SEQ ID NO:9 with SEQ IDNO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, the coded sequence of SEQ ID NO:8 or SEQ ID NO:9 has at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity again.At this on the one hand, highly preferably have the polynucleotide of at least 97% homogeneity, and more highly preferably have at least polynucleotide of 98-99% homogeneity, topnotch preferably has the polynucleotide of at least 99% homogeneity.Such polynucleotide comprise containing SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, the polynucleotide of SEQ ID NO:8 or SEQ ID NO:9 polynucleotide and SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, the polynucleotide of SEQ ID NO:9 or SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, the coding region of SEQ ID NO:8 or SEQ ID NO:9.

The present invention also provides the nucleic acid of coding aforementioned foreign protein of the present invention and the application at field of medicaments thereof.In a preferred embodiment, be shown in SEQ ID NO:13 (mice) or SEQ ID NO:15 (rat) for the sequence of the xenogenesis CASB7439 polynucleotide of Pharmaceutical composition.Can be strand (coding strand or antisense strand) or two strands according to the xenogenesis CASB7439 polynucleotide of separation of the present invention, and can be DNA molecular (genome molecule, cDNA molecule or synthetic molecules) or RNA molecule.Other coded sequence or non-coding sequence can but be not necessarily present in polynucleotide of the present invention.In other related embodiment, the invention provides polynucleotide variant, in described polynucleotide variant and SEQ ID NO:13 or SEQ ID NO:15, sequence disclosed herein has roughly homogeneity, for example, adopt the method as herein described BLAST of canonical parameter (for example use analyze) to comprise at least 70% sequence homogeneity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or the polynucleotide variant of higher sequence homogeneity compared with polynucleotide sequence of the present invention.In a related embodiment, the xenogenesis polynucleotide of separation of the present invention comprise in the total length that is coded in SEQ ID NO:12 or SEQID NO:14 with the aminoacid sequence of SEQ ID NO:12 or SEQ ID NO:14 have at least 90%, preferably 95% with the nucleotide sequence of the polypeptide of 95% above homogeneity or with the nucleotide sequence of the described polynucleotide complementation separating.

The present invention also provides the polynucleotide with all above-mentioned polynucleotide complementations.

In the recombinant microorganism that described polynucleotide can be inserted into suitable plasmid, recombinant microorganism carrier or live, and can use for immunity inoculation (referring to such as Wolff etc., Science247:1465-1468 (1990); Corr etc., J.Exp.Med.184:1555-1560 (1996); Doe etc., Proc.Natl.Acad.Sci.93:8578-8583 (1996)).Therefore, the invention provides and comprise expression vector or the recombinant microorganism alive of described polynucleotide as defined above.

The present invention also provides the fragment of CASB7439 polynucleotide, wherein in the time giving curee described in the immunogenicity characteristic of fragment identical with the immunogenicity characteristic of following polynucleotide: SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:15.

The present invention also provides the polynucleotide of the immunity fragment of coding as previously defined CASB7439 polypeptide.

The immunogenicity activity level of described fragment is SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10 or SEQ ID NO:11, peptide sequence shown in SEQ ID NO:12 or SEQID NO:14 or by SEQ ID NO:1, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, the immunogenicity activity level of the peptide sequence of the polynucleotide sequence coding shown in SEQ ID NO:13 or SEQ ID NO:15 at least about 50%, preferably at least about 70%, more preferably at least about 90%.

Preferably comprise at least about 5 according to polypeptide fragment of the present invention, 10, 15, 20, 25, 50 or 100 continuous amino acids or 100 above continuous amino acids, comprise the peptide composition shown in this article of all length placed in the middle, for example SEQ ID NO:2, SEQID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, peptide composition shown in SEQ ID NO:12 or SEQ ID NO:14, or by SEQ ID NO:1, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, the peptide composition of the polynucleotide sequence coding shown in the sequence of SEQ ID NO:13 or SEQ ID NO:15.

The nucleotide sequence of SEQ ID NO:1 is the cDNA sequence that comprises the polypeptid coding sequence (nucleotide 545-1126) that 193 amino acid whose polypeptide of coding are the polypeptide of SEQ ID NO:2.The nucleotide sequence of the polypeptide of coding SEQ ID NO:2 can be identical with polypeptid coding sequence contained in SEQ ID NO:1, or it can be the sequence that is different from polypeptid coding sequence contained in SEQ ID NO:1, due to the Feng Yuxing (degeneracy) of genetic code, also the encode polypeptide of SEQ ID NO:2 of described nucleotide sequence.The polypeptide of SEQ ID NO:2 is structurally relevant to other albumen of achaete scute family, and also called after " people Achaete Scute congener 2 " (HASH2) (registration number NP_005161 and AAB86993).

People Achaete Scute congener 2 (HASH2) gene-definite designation behaviour ASCL2 (Achaete Scute complex sample 2) is the congener of Drosophila (Drosophila) Achaete and Scute gene.People ASCL2 only expresses in fine hair (extravillus trophoblast) outside the trophoderm of developmental Placenta Hominis, and it is upper near IGF2 and H19 to chromosome 11p15 to map.Mice achaete-scute homologue-2 genes (MASH2) are coded in the transcription factor working in trophoblastic growth.Mash2 gene is paternal inheritance in mice, and in non-malignant blister shape (gynogenesis (andorgenetic)) mole, lacks the expression of people ASCL2, shows also heredity in the mankind of people Ascl2.

Ascl2 gene is the member of alkaline helix-loop-helix (BHLH) family of transcription factor.They by with E frame (5 '-CANNTG-3 ') in conjunction with and activated transcription.It is Dimerized with other BHLH albumen that to be that effective dna is combined required.They are at Drosophila melanogaster (Drosophilamelanogaster) and may in mammal, participate in the neuron precursor that determines that peripheral nervous system is unified in central nervous system.

The complementary strand of SEQ ID NO:1 nucleotide sequence is the polynucleotide sequence of SEQ ID NO:6.This chain also comprises two kinds of other polypeptid coding sequences.The first polypeptid coding sequence (the nucleotide 1184-399 of SEQ IDNO:1, the nucleotide 608-1393 of SEQ ID NO:6) coding a kind of 262 amino acid whose polypeptide, the i.e. polypeptide of SEQ ID NO:3.The second polypeptid coding sequence (the nucleotide 840-262 of SEQ ID NO:1, the nucleotide 952-1530 of SEQ ID NO:6) coding a kind of 193 amino acid whose polypeptide, the i.e. polypeptide of SEQ ID NO:11.The nucleotide sequence of coding SEQ IDNO:3 and the polypeptide of SEQ ID NO:11 can be identical with polypeptid coding sequence contained in SEQ ID NO:6, or it can be the sequence that is different from polypeptid coding sequence contained in SEQ ID NO:6, due to the Feng Yuxing (degeneracy) of genetic code, also the encode polypeptide of SEQ ID NO:3 and SEQ ID NO:11 of described nucleotide sequence.The polypeptide of SEQ IDNO:3 is structurally relevant to other albumen of montage coactivator family, has homology and/or structural similarity with people (Homo sapiens) montage coactivator subunit srm300 (Genbank registration number AAF21439).The polypeptide of SEQ ID NO:11 is uncorrelated with any known albumen.Polynucleotide sequence shown in peptide sequence shown in SEQ ID NO:3 and SEQ ID NO:11 and SEQ ID NO:6 is new sequence, and also forms a part of the present invention.

Expect preferred polypeptide of the present invention and polynucleotide inter alia, similar to the biological function/characteristic of its homeopeptide and polynucleotide.In addition, the preferred polypeptide of the present invention, immunity fragment and polynucleotide have at least one suitable activity of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:11.

The invention still further relates to part or other incomplete polynucleotide sequence and peptide sequence, described sequence is first to identify before the corresponding full length sequence of measuring SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ IDNO:11.

, more on the one hand, the invention provides the polynucleotide of separation, described polynucleotide therefore:

(a) be included in the total length of SEQ ID NO:4 and SEQ ID NO:5 with SEQ ID NO:4 and SEQ ID NO:5 and there is at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity, the even more preferably nucleotide sequence of 97-99% homogeneity at least again;

(b) have in the total length of SEQ ID NO:4 and SEQ ID NO:5 respectively with SEQ IDNO:1 or SEQ ID NO:6 and there is at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity, the even more preferably nucleotide sequence of 97-99% homogeneity at least again;

(c) polynucleotide of SEQ ID NO:4 and SEQ ID NO:5; Or

(d) polynucleotide of the nucleotide sequence of coded polypeptide and SEQ ID NO:4 and SEQ ID NO:5, described polypeptide has at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity, even more preferably 97-99% homogeneity at least again with the aminoacid sequence of SEQ ID NO:2 and SEQ ID NO:7 respectively in the total length of SEQ ID NO:2 and SEQ ID NO:7.

The present invention also provides such polypeptide, described polypeptide:

(a) be included in the total length of SEQ ID NO:2 or SEQ ID NO:7 with the aminoacid sequence of SEQ ID NO:2 and SEQ ID NO:7 and there is at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity, the aminoacid sequence of 97-99% homogeneity at least most preferably again;

(b) have in the total length of SEQ ID NO:2 or SEQ ID NO:7 with the aminoacid sequence of SEQ ID NO:2 or SEQ ID NO:7 and there is at least 70% homogeneity, preferably at least 80% homogeneity, more preferably at least 90% homogeneity, more preferably at least 95% homogeneity, the aminoacid sequence of 97-99% homogeneity at least most preferably again;

(c) aminoacid that comprises SEQ ID NO:2 or SEQ ID NO:7; With

(d) be the polypeptide of SEQ ID NO:7;

And the present invention also provides by the polypeptide of polynucleotide encoding that is included in sequence contained in SEQ ID NO:4 and SEQ ID NO:5.

Can employing standard clone and triage techniques, in the derivative cDNA library of mRNA from human colon cancer cell, obtain polynucleotide of the present invention, (such as Sambrook etc., MolecularCloning:A Laboratory Manual, the second edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989)).Polynucleotide of the present invention also can derive from the natural origin such as genome dna library, or can adopt well-known commercially obtainable technology to synthesize.

When with polynucleotide recombinant production of the present invention polypeptide of the present invention, described polynucleotide can comprise the coded sequence of mature polypeptide itself; Or in frame, also has for example the encode sequence of targeting sequencing or secretion sequence, front albumen (pre-protein) sequence or former albumen (pro-protein) sequence or front former albumen (prepro-protein) sequence or other fusogenic peptide part of the coded sequence of mature polypeptide of other coded sequence, described other coded sequence.For example, can encode and be conducive to the labelled sequence of the polypeptide that purification merges.In some preferred embodiment in the present invention aspect this, described labelled sequence is at pQE carrier (Qiagen, Inc.) in, provide and at Gentz etc., the six histidine peptides of describing in Proc Natl Acad Sci USA (1989) 86:821-824, or HA labelling.Described polynucleotide also can contain 5 ' and 3 ' non-coding sequence, for example, transcribe but the sequence of sequence, splicing signal and polyadenylation signal, ribosome binding site and the stable mRNA do not translated.

Other embodiment of the present invention comprises the polynucleotide of coded polypeptide variant, the aminoacid sequence that described polypeptide variants comprises SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ IDNO:11, SEQ ID NO:13 or SEQ ID NO:15, wherein several, for example 5-10,1-5,1-3,1-2 or 1 aminoacid replaces, lacks or add with any combination.

Identical with nucleotide sequence contained in SEQ ID NO:1 or SEQ ID NO:6 or enough identical polynucleotide can be as the hybridization probe of cDNA and genomic DNA or as the primer of nucleic acid amplification (PCR) reaction, to separate full-length cDNA and the genomic clone of code book invention polypeptide, and separation and SEQ ID NO:1 or SEQ ID NO:6 have cDNA and the genomic clone of other gene (comprising that coding derives from people's symbiosis congener and derives from the straight homologues of the species except the mankind and the gene of symbiosis congener) of height sequence similarity.Conventionally, these nucleotide sequences and the sequence of reference substance have 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical.Described probe or primer generally comprise at least 15 nucleotide, at least 30 nucleotide preferably, and can have at least 50 nucleotide.Particularly preferred probe has 30-50 nucleotide.Particularly preferred primer has 20-25 nucleotide.Specifically, come from allogenic animal polypeptide or polynucleotide that source sequence obtains and can be used as immunogen, to obtain the immunne response that has cross reaction with people's gene.

The polynucleotide of code book invention polypeptide including the homologue of the species from except people, can obtain by such method: described method is included under stringent hybridization condition, with thering is SEQ ID NO:1 or the sequence of SEQ ID NO:6 or the label probe of its fragment, screen suitable library; And the full-length cDNA that separation contains described polynucleotide sequence and the step of genomic clone.Such hybridization technique is well known to the skilled person.Preferred stringent hybridization condition is included in the solution that comprises following component in 42 ℃ of incubated overnight: the salmon sperm DNA that 50% Methanamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x DenhardtShi solution, 10% dextran sulfate and 20 micrograms/ml degeneration are sheared; Then in 0.1x SSC, at approximately 65 ℃, wash filter membrane.Therefore, the present invention is also included within stringent hybridization condition, with having SEQ ID NO:1 or the sequence of SEQ ID NO:6 or the label probe of its fragment, by screening the obtainable polynucleotide in suitable library.

Technical staff knows, in many cases, the cDNA sequence of separation is incomplete, because the coding region of described polypeptide is shorter at the 5 ' end of described cDNA.

To those skilled in the art, the method that has several to obtain full-length cDNAs or to extend short cDNA can be utilized and be well-known, for example those methods based on cDNA end rapid amplifying (RACE) method are (referring to such as Frohman etc., PNAS USA 85,8998-9002,1988).For example, with Marathon ^tMup-to-date improved this technology that technology (Clontech Laboratories Inc.) is example has been simplified the search to longer cDNA greatly.At Marathon ^tMin technology, use the mRNA extracting from selected tissue to prepare cDNA, and " joint " sequence is connected to each end.Then use the combination of gene specific oligonucleotide primers and joint specific oligonucleotide primer, carry out nucleic acid amplification (PCR), with described cDNA " disappearance " 5 ' end that increases.Then use " nested " primer to repeat described PCR reaction, " nested " primer is the primer (by being the joint Auele Specific Primer of 3 ' annealing and the gene-specific primer of 5 ' farther annealing in known gene order farther in joint sequence) that design is annealed in increased product.Then can analyze by DNA sequencing the product of this reaction, produce complete sequence by making described product be directly connected to existing cDNA subsequently, or use new sequence information design 5 ' primer to carry out an independently total length PCR, build full-length cDNA.

By method well-known in the art, can prepare recombinant polypeptide of the present invention with the genetically engineered host cell that comprises expression system., more on the one hand therefore, the present invention relates to the expression system that comprises polynucleotide of the present invention, relate to the host cell genetically engineered with such expression system, also relate to by recombinant technique and produce polypeptide of the present invention.Also can adopt the RNA derived from DNA construct of the present invention, use cell free translation system to produce such protein.

For recombinant production, can genetically engineered host cell to mix expression system or its part of polynucleotide of the present invention.Can adopt the method for introducing in many standard laboratory handbooks, realize polynucleotide are imported to host cell, such as Davis of described laboratory manual etc., Basic Methods in Molecular Biology (1986) and Sambrook etc., MolecularCloning:A Laboratory Manual, the second edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).Preferred these class methods comprise transfection, electroporation, transduction, scratch application of sample (scrape loading), the particle gun introducing (ballisticintroduction) that for example calcium phosphate transfection, the transfection of DEAE-glucosan mediation, transposition, microinjection, cation lipid mediate or infect.

Albumen of the present invention preferably with trans thioredoxin (TIT) coexpression.Preferably trans and cis thioredoxin coexpression, to keep antigen without thioredoxin, and does not need protease.Thioredoxin coexpression makes albumen of the present invention be easy to dissolve.Thioredoxin coexpression is also on protein purification yield, dissolubility and mass formation appreciable impact on purifying protein.

The representative example of suitable host comprises bacterial cell, as streptococcus (Streptococci), staphylococcus (Staphylococci), escherichia coli, streptomyces (Streptomyces) and bacillus subtilis (Bacillus subtilis) cell; Fungal cell, the cell of for example yeast cells and aspergillus (Aspergillus); Insect cell, for example Drosophila (Drosophila) S2 cell and greedy noctuid (Spodoptera) Sf9 cell; Zooblast, for example CHO, COS, HeLa, C127,3T3, BHK, HEK 293 and Bowes melanoma cells; And plant cell.

Can use various expression systems, derivative system and the derivative system of virus of system, episome that for example chromosome is derivative, for example, derived from following carrier: bacterial plasmid, phage, transposon, yeast episome, insertion element, yeast chromosomal element; Carrier derived from following virus: baculovirus, papovavirus (as SV40), vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus retrovirus; And derived from the carrier of its combination, for example, derived from those carriers of plasmid and phage genetic elements, such as cosmid and phasmid.Described expression system can comprise adjusting and produce the control zone of expressing.Generally speaking, can use and can in host, maintain, breed or express polynucleotide to produce any system or the carrier of polypeptide.Can be by multiple well-known routine techniques any, suitable nucleotide sequence is inserted in expression system, such as Sambrook of described routine techniques etc., the technology that Molecular Cloning:A Laboratory Manual (referring to above) introduces.In order to allow the protein excretion of translation in endoplasmic, periplasmic space or born of the same parents' external environment, suitable secretion signal can be incorporated in required polypeptide.These signals can be endogenous for described polypeptide, or they can be allos signals.

Described expression system can be also the recombinant microorganism of living, for example virus or antibacterial.Interested gene can be inserted in the genome of recombinant virus alive or antibacterial.With this carrier inoculation and In vivo infection of living, will make described antigen express in vivo and induce immune response.

Therefore, in certain embodiments, adopt the arbitrary system in numerous known systems based on viral, the polynucleotide of code book invention immunogenic polypeptide are imported to the suitable mammalian host cell for expressing.In an illustrative embodiment, retrovirus retrovirus is provided for the effective platform of facility of gene delivery system.Adopt technology known in the art, the selected nucleotide sequence of code book invention polypeptide can be inserted in carrier, then at counter-transcription-ing virus particle intermediate package.Then can separate described recombinant virus, be given curee.The existing introduction of many illustrative retrovirus retrovirus systems (for example United States Patent (USP) the 5th, 219, No. 740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A.D. (1990) Human Gene Therapy 1:5-14; Scarpa etc. (1991) Virology 180:849-852; Burns etc. (1993) Proc.Natl.Acad.Sci.USA 90:8033-8037; With Boris-Lawrie and Temin (1993) Cur.Opin.Genet.Develop.3:102-109).

In addition, many illustrative systems based on adenovirus also have introduction.Retrovirus retrovirus in host genome is different from being incorporated into, and adenovirus is positioned at outside chromosome always, thereby the danger relevant to inserting mutation is reduced to minimum (Haj-Ahmad and Graham (1986) J.Virol.57:267-274; Bett etc. (1993) J.Virol.67:5911-5921; Mittereder etc. (1994) Human Gene Therapy 5:717-729; Seth etc. (1994) J.Virol.68:933-940; Barr etc. (1994) Gene Therapy 1:51-58; Berkner, K.L. (1988) BioTechniques6:616-629; With (1993) Human Gene Therapy 4:461-476 such as Rich).

Also develop various adeno associated viruss (AAV) carrier system in order to transmit polynucleotide.Adopt technology well-known in the art, can easily build AAV carrier.Referring to, for example, United States Patent (USP) the 5th, 173, No. 414 and the 5th, 139, No. 941; International publication number WO 92/01070 and WO 93/03769; Lebkowski etc. (1988) Molec.Cell.Biol.8:3988-3996; Vincent etc. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B.J. (1992) Current Opinion in Biotechnology3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol.and Immunol.158:97-129; Kotin, R.M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; With (1994) J.Exp.Med.179:1867-1875 such as Zhou.

Can be used for passing by gene other viral vector that moves the nucleic acid molecules that transmits code book invention polypeptide and comprise for example, viral vector derived from Poxviridae (vaccinia virus and fowlpox virus).As an example, recruit's vaccinia virus recombinant described in construction expression as follows.First the DNA of coded polypeptide is inserted in suitable carrier, makes it for example, in abutting connection with a vaccinia virus promoter and flank vaccinia virus DNA sequence, the sequence of thymidine kinase (TK) that encode.Then use the cell of vaccinia virus infection with this carrier transfection simultaneously.Homologous recombination makes vaccinia virus promoter add that the gene of the interested polypeptide of coding is inserted in viral genome.By leave lower cultured cell at 5-bromouracil deoxyribose, then choose resistance virus plaque, can select produced TK.sup. (-) recombinant.

Can use easily infection/transfection system based on vaccinia virus that induction type transient expression or the coexpression of one or more polypeptide as herein described are provided in the host cell of a certain biology.In this particular system, use first in vitro the vaccinia virus recombinant infection cell of coding phage t7 RNA polymerase.This polymerase shows sensitive specificity, because it only transcribes the template with T7 promoter.After infection, cell one or more interested polynucleotide transfections by T7 promoters driven.The DNA of transfection is transcribed into RNA by the polymerase of expressing in kytoplasm from vaccinia virus recombinant, then translated into polypeptide by host's machine translator.Described method is available for high level, instantaneous, a large amount of RNA and the translation products thereof of the interior production of kytoplasm.Referring to, for example, Elroy-Stein and Moss, Proc.Natl.Acad.Sci.USA (1990) 87:6743-6747; Fuerst etc., Proc.Natl.Acad.Sci.USA (1986) 83; 8122-8126.

Or, also can use such as the fowlpox virus (avipoxvirus) of fowlpox virus (fowlpox virus) and canary pox virus and transmit interested coded sequence.The immunogenic recombinant fowlpox virus (avipox virus) that known expression derives from mammal pathogen provides protective immunity in the time giving non-avian species.It is desirable especially in people and other mammalian species, applying fowlpox virus carrier, and this is because the member of Avipoxvirus only can productively copy in the avian species of susceptible, therefore they in mammalian cell, be do not have infectious.The method of preparing recombinant fowlpox virus is known in this area, and uses the genetic recombination about preparation vaccinia virus as above.Referring to, for example, WO91/12882; WO89/03429 and WO92/03545.

Can also transmit polynucleotide compositions of the present invention with any in numerous Alphavirus carriers, for example, at U.S. Patent number 5,843, those carriers of introducing in 723,6,015,686,6,008,035 and 6,015,694.Also can use some carrier based on Venezuelan equine encephalitis (VEE) virus, its illustrative example can be at United States Patent (USP) the 5th, 505, No. 947 and the 5th, finds in 643, No. 576.

In addition, can also use the molecule such as adenovirus chimeric vector of introducing in Proc.Natl.Acad.Sci.USA (1992) 89:6099-6103 such as the J.Biol.Chem. such as Michael (1993) 268:6866-6869 and Wagner to put together carrier and carry out gene delivery of the present invention.

Other accountability information of relevant these and other known transmission system based on viral can find in following document: such as Fisher-Hoch etc., Proc.Natl.Acad.Sci.USA86:317-321,1989; Flexner etc., Ann.N.Y.Acad.Sci.569:86-103,1989; Flexner etc., Vaccine 8:17-21,1990; U.S. Patent number 4,603,112,4,769,330 and 5,017,487; WO 89/01973; U.S. Patent number 4,777,127; GB 2,200,651; EP0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627,1988; Rosenfeld etc., Science 252:431-434,1991; Kolls etc., Proc.Natl.Acad.Sci.USA 91:215-219,1994; Kass-Eisler etc., Proc.Natl.Acad.Sci.USA90:11498-11502,1993; Guzman etc., Circulation 88:2838-2848,1993; With Guzman etc., Cir.Res.73:1202-1207,1993.

The recombinant microorganism of above-mentioned work can have virulence, or in every way attenuation with obtain live vaccine.Such live vaccine also forms a part of the present invention.

In certain embodiments, polynucleotide can be incorporated in the genome of target cell.This integration can be carried out (gene substitution) with specific direction by homologous recombination at ad-hoc location, or it can integrate (gene amplification) in random, nonspecific position.In other embodiment, described polynucleotide can be in cell stably keep as additive type DNA section independently.The sequence of such polynucleotide section or " episome " coding is enough to allow its maintenance and is independent of that the host cell cycle copies or copies with host cell cycle synchronisation.Mode and described polynucleotide that expression construct is passed to cell remain on the type that expression construct used is depended in described intracellular position.

In another embodiment of the present invention, for example, as at Ulmer etc., Science259:1745-1749, description and Cohen in 1993, Science 259:1691-1692,1993 summary, polynucleotide give/transmit as " naked " DNA.By naked DNA being coated with by the biodegradable microballon in transporte to cells effectively, can increase the absorption of described DNA.

In an embodiment again, compositions of the present invention can be transmitted by Particle bombardment, and wherein many methods are existing introduces.In an illustrative embodiment, use such as PowderjectPharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc. (Madison, WI) device of producing can be realized pneumatic particle and accelerate, wherein some embodiment is described in U.S. Patent number 5,846, and 796,6,010,478,5,865,796,5,584,807 and european patent number 0500799.The method provides a kind of needleless transmission method, is wherein accelerated at a high speed in the helium injection stream being produced by hand held device such as the microgranule dry powder formulations of polynucleotide or polypeptide particle, promotes described particle and arrives interested target tissue.

In a related embodiment, can comprise Bioject for other apparatus and method of the pneumatic Needleless injection present composition, the apparatus and method that Inc. (Portland, OR) provides, wherein some embodiment is described in U.S. Patent number 4,790, and 824,5,064,413,5,312,335,5,383,851,5,399,163,5,520,639 and 5,993,412.

By well-known method, can from recombinant cell culture thing, reclaim and purification polypeptide of the present invention, described method comprises ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion-exchange chromatography or cation-exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatograph, hydroxyapatite chromatography and agglutinin chromatography.Most preferably use ionic metal affinity chromatograph (IMAC) to carry out purification.When degeneration, can use the refolding albumen reconstruction activity conformation of knowing when described polypeptide is synthetic in born of the same parents, in separation and/or purge process.

Another importance of the present invention relates to induction, strengthens or regulate the method for immunne response in mammalian body, described method comprises with being enough to produce of the present invention complete polypeptide or polynucleotide or its fragment seeded with mammalian of antibody and/or T cellullar immunologic response, for immunoprophylaxis or being used for the treatment of property treatment cancer, more especially colorectal carcinoma and autoimmune disease and associated conditions.The method that relates in one aspect to again induction, strengthens or regulate immunne response in mammalian body of the present invention, described method comprises that carrier or the cell of expressing described polynucleotide and coding said polypeptide by instructing in vivo transmit polypeptide of the present invention, to induce such immunne response, to produce prevention or to treat described animal exempt from the to take a disease immunne response of disease.

Of the present inventionly relate in one aspect to again immune formulation/bacterin preparation (compositions) and the application at field of medicaments thereof.When described compositions is introduced into after mammalian hosts, induction in this mammalian body, reinforcement or the metering needle immunne response to polypeptide of the present invention, wherein said compositions comprises the polypeptide of the present invention or polynucleotide or its immunology fragment that are limited as herein above.More particularly, the CASB7439 polypeptide that comprises safe and effective amount according to immunogenic composition of the present invention or its immunogenic fragments, wherein said CASB7439 polypeptide is selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12 or SEQ ID NO:14.In another embodiment, the polynucleotide of the coding CASB7439 that described immunogenic composition comprises safe and effective amount or its fragment, the polynucleotide of wherein said coding CASB7439 are selected from SEQ ID NO:1, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:15.

Can also comprise a kind of suitable carrier, i.e. pharmaceutically acceptable carrier according to bacterin preparation of the present invention.Because polypeptide may decompose under one's belt, therefore they preferably parenteral give (for example subcutaneous injection, intramuscular injection, intravenous injection or intradermal injection).The preparation that is suitable for parenteral comprises aqueous and non-water aseptic parenteral solution, the solute that described injection can comprise antioxidant, buffer agent, antibacterial and described preparation and receptor blood etc. are oozed; And can comprise aqueous and the non-water sterile suspensions of suspending agent or thickening agent.Described preparation for example can be contained in, in unit-dose container or multi-dose container (sealed ampoule and phial), and can under lyophilisation condition, preserve, and only need to add before use sterile liquid carrier.

The cell adopting from immune system that relates in one aspect to again of the present invention, induce in vitro for the immunne response of complete polypeptide of the present invention or polynucleotide or its fragment or for the immunne response of the molecule that comprises polypeptide of the present invention or polynucleotide, and these activating immune cells are fed back to described mammal with treatment disease.By with the complete polypeptide of the present invention or polynucleotide or the molecule that comprises polypeptide of the present invention or polynucleotide having or incubated in vitro without various immune modulatory molecules in the situation that, reach activation from described immune described cell.Of the present invention relating in one aspect to again by giving antigen-presenting cell immunity mammal, described antigen-presenting cell is modified by the molecule that loads in vitro complete polypeptide of the present invention or its part or comprise polypeptide of the present invention, then in immunogenicity mode by described antigen-presenting cell donor.Or, can be with the carrier of the molecule that contains complete polynucleotide of the present invention or its fragment or comprise polynucleotide of the present invention, in-vitro transfection antigen-presenting cell, for example, to express corresponding polypeptide, then in immunogenicity mode by described antigen-presenting cell donor.Therefore, the antigen-presenting cell that Pharmaceutical composition of the present invention comprises effective dose and pharmaceutically effective carrier, described antigen-presenting cell is modified by loading in vitro CASB7439 polypeptide or the genetically modified CASB7439 of expression polypeptide in vitro.

According to another embodiment, except immunogenicity polynucleotide of the present invention, polypeptide, antibody, T cell and/antigen-presenting cell (APC) compositions, Pharmaceutical composition/immunogenic composition as herein described also comprises one or more immunostimulant.Therefore, provide the method for producing described immunogenic composition herein, described method comprises mixes the polynucleotide of CASB7439 polypeptide or coding CASB7439 with suitable adjuvant/immunostimulant, diluent or other pharmaceutically acceptable carrier.Immunostimulant refers in fact any material of the immunne response that improves or strengthen exogenous antigen (antibody and/cell-mediated).A kind of immunostimulant of preferred type comprises adjuvant.Many adjuvants contain design in order to protect antigen to exempt from the material of fast decoupled; for example aluminium hydroxide or mineral oil; and containing immune stimulant; for example lipid A, Bordetella pertussis (Bortadella pertussis) or mycobacterium tuberculosis (Mycobacterium tuberculosis) endogenous binding protein.Some adjuvant is commercially available, for example incomplete Freund's adjuvant and Freund's complete adjuvant (Difco Laboratories, Detroit, MI); Merck adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKlineBeecham, Philadelphia, PA); Such as the aluminum salt of gel aluminum hydroxide (Alumen) or aluminum phosphate; Calcium salt, iron salt or zinc salt; The insoluble suspending agent of acidylate tyrosine; Acidylate sugar; The polysaccharide of cation or anionic derivative; Polyphosphazene; Biodegradable microsphere; Single phosphatidyl lipid A and quil A.Cytokine and other similar somatomedin such as GM-CSF, interleukin-2, IL-7, IL-12 also can be used as adjuvant.

In certain embodiments of the invention, described adjunvant composition is preferably mainly induced the adjunvant composition of Th1 type immunne response.High-caliber Th1 cytokines (for example IFN-γ, TNF α, IL-2 and IL-12) often promotes the cell-mediated immunne response of induction for given antigen.On the contrary, high-caliber Th2 cytokines (for example IL-4, IL-5, IL-6 and IL-10) often promotes induction humoral immunoresponse(HI).Apply after vaccine provided herein, patient will support to comprise the immunne response that Th1 type and Th2 type are replied.Replying in a preferred embodiment that is mainly Th1 type, the degree that the level of Th1 cytokines increases than the level of Th2 cytokines is larger.Adopt standard test can easily evaluate the level of these cytokines.The summary of cells involved factor family, refers to Mosmann and Coffman, Ann.Rev.Immunol.7:145-173,1989.

For induction be mainly some preferred adjuvant that Th1 type replys for example comprise single phosphatidyl lipid A, preferably 3-de--combination of O-acidylate list phosphatidyl lipid A together with aluminum salt. adjuvant can derive from Corixa Corporation (Seattle, WA; Referring to, for example, U.S. Patent number 4,436,727,4,877,611,4,866,034 and 4,912,094).Oligonucleotide (wherein CpG dinucleotide is not methylated) containing CpG is also induced and is mainly replying of Th1 type.Such oligonucleotide is well-known, is described in for example WO 96/02555, WO99/33488 and U.S. Patent number 6,008,200 and 5,856,642.Immunostimulation DNA sequence is also described in such as Sato etc., Science 273:352,1996.Another preferred adjuvant comprises the saponin or derivatives thereof such as Quil A, comprises QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA); Aescine; Digitonin; Or chalk-plant (Gypsophila) or Chenopodium quinoa saponin.In adjuvant combination of the present invention, other preferred preparation comprises more than one saponin, for example the combination of following at least two kinds: QS21, QS7, QuilA, β-aescine or digitonin.

Or, saponin preparation can be mixed with the vaccine carrier forming by chitosan or other polycationic polymer, polyactide and PLGA granule, polymeric matrix based on poly-n-acetyl glycosamine, by polysaccharide or through granule, liposome and the granule based on lipid, the granule being made up of monoglyceride etc. of the composition of Salvia polysaccharide of chemical modification.Saponin also can be prepared under cholesterol exists, for example, to form graininess structure, liposome or ISCOM.In addition, saponin can be together with polyoxyethylene ether or ester at non-particulate property solution or suspension or prepare in the graininess structure such as paucilamelar liposome or ISCOM.Saponin also can with such as Carbopol ^rexcipient prepare together, to increase viscosity, or can prepare with together with powdery excipient such as lactose with dry powder form.

In a preferred embodiment, described adjuvant system comprises the combination of single phosphatidyl lipid A and saponin derivative, for example in WO 94/00153 describe QS21 and

the combination of adjuvant or the wherein QS21 describing in the WO 96/33739 lower compositions of reactionogenicity of cholesterol quencher.Other preferred preparation comprises oil in water emulsion and tocopherol.In WO 95/17210, described use QS21, another particularly preferred adjuvant formulation of the oil in water emulsion of adjuvant and tocopherol.

The adjuvant system of another enhancing comprises the combination containing disclosed CpG and QS21 in combination, the especially WO 00/09159 of CpG ODN and saponin derivative.Described preparation preferably comprises oil in water emulsion and tocopherol in addition.

Other illustrative adjuvant being available in Pharmaceutical composition of the present invention comprises MontanideISA 720 (Seppic, France), SAF (Chiron, California, the U.S.), adjuvant (for example SBAS-2 or the SBAS-4 of ISCOMS (CSL), MF-59 (Chiron), SBAS series, can derive from SmithKline Beecham, Rixensart, Belgium), Detox

(Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphate ester (AGP), the U.S. Patent application serial number 08/853,826 and 09/074 of for example pending trial, adjuvant (disclosure of described patent application is all attached to herein by reference) and the polyoxyethylene ether adjuvant in 720, described, the adjuvant of for example describing in WO 99/52549A1.

Other preferred adjuvant comprises the adjuvant molecules of general formula (I):

HO(CH ₂CH ₂O) _n-A-R

Wherein n is 1-50, A be a key or-C (O)-, R is C _1-50alkyl or phenyl C _1-50alkyl.

One embodiment of the invention comprise that wherein n is 1-50, is preferably 4-24, most preferably is 9 containing the bacterin preparation of the polyoxyethylene ether of general formula (I); R component is C _1-50alkyl, be preferably C _4-20alkyl, most preferably be C ₁₂alkyl; A is a key.The concentration range of polyoxyethylene ether should be 0.1-20%, is preferably 0.1-10%, most preferably is 0.1-1%.Preferred polyoxyethylene ether is selected from following group: Polyoxyethylene-9-lauryl ether, polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether and polyoxyethylene-23-lauryl ether.Polyoxyethylene ether such as polyoxyethylene lauryl ether has description in Merck index (the 12nd edition: entry 7717).These adjuvant molecules have description in WO99/52549.

If need, according to the polyoxyethylene ether of above-mentioned general formula (I) can with another adjuvant combination.For example a kind of preferred adjuvant combination preferably with the UK Patent Application GB 9820956.2 of pending trial in the CpG combination described.

According to preferably also there being a kind of carrier in vaccine combination of the present invention.Described carrier can be oil in water emulsion, or a kind of aluminum salt, for example aluminum phosphate or aluminium hydroxide.

Preferred oil in water emulsion comprises a kind of metabolizable oil, for example Squalene, alpha-tocopherol and Tween 80.One particularly preferred aspect, mix with QS21 and 3D-MPL in this Emulsion according to the antigen in vaccine combination of the present invention.In addition, described oil in water emulsion can contain span 85 and/or lecithin and/or tricaprylin.

While giving people, conventionally the QS21 in vaccine and 3D-MPL content range are every dose of 1 μ g-200 μ g, for example 10 μ g-100 μ g, 10 μ g-50 μ g preferably.And oil in water emulsion comprises 2-10% Squalene, 2-10% alpha-tocopherol and 0.3-3% Tween 80 conventionally.Squalene: the ratio of alpha-tocopherol is preferably equal to or less than 1, because such ratio provides more stable Emulsion.The content of Span 85 can be also 1%.In some cases, to contain in addition a kind of stabilizing agent may be favourable to vaccine of the present invention.

Nontoxic oil in water emulsion is preferably in and in aqueous carrier, contains for example squalane of a kind of nontoxic oil or Squalene and such as Tween 80 of a kind of emulsifying agent.Described aqueous carrier can be for example phosphate buffered saline(PBS).

In WO 95/17210, described especially effectively adjuvant formulation of one, said preparation is the oil in water emulsion that comprises QS21,3D-MPL and tocopherol.

The present invention also provides multivalent vaccine composition, and described multivalent vaccine composition comprises bacterin preparation of the present invention and other antigen, is particularly useful for treating cancer, the antigen of colorectal carcinoma, autoimmune disease and associated conditions more especially.Such multivalent vaccine composition can comprise TH-1 inducing adjuvant as described above.

According to another embodiment of the invention, immunogenic composition as herein described is passed to host by antigen-presenting cell (APC), and described antigen-presenting cell (APC) for example dendritic cell, macrophage, B cell, mononuclear cell and other can be by engineered to become the cell of effective APC.This class cell can but not necessarily by genetic modification, with increase antigen-presenting ability, improve t cell response activation and/or maintenance, itself there is Graft Versus Tumor and/or in immunology, be compatible (mating HLA haplotype) with receptor.APC generally can separate from any of various biological fluids or organ (comprising tumor and tumor surrounding tissue), can be maybe autogenous cell, homogeneous variant cell, homogenic cell of the same race or heterogenous cell.

Some preferred embodiment of the present invention is used dendritic cell or its CFU-GM as antigen-presenting cell.Dendritic cell are very effective APC (Banchereau and Steinman, Nature392:245-251,1998), and show that as induction physiology adjuvant preventative or that therapeutic anti-tumour is immune be effectively (referring to Timmerman and Levy, Ann.Rev.Med.50:507-529,1999).Generally speaking, dendritic cell may according to its typical shape (original position is starlike, external visible significantly cytoplasmic process (dendron)), its absorption, projection and with the ability of high efficiency antigen-presenting with and the originally ability of t cell response that activates identify.Certainly, can carry out dendritic cell engineered, with express conventionally in vivo or in vitro dendritic cell on non-existent specific cell surface receptor or part, the present invention has considered the modified dendritic cell of this class.As the substitute of dendritic cell, in vaccine, can use the dendritic cell (being called allochthon) (referring to Zitvogel etc., Nature Med 4.594-600,1998) that load secreting type vesicle antigen.

Dendritic cell and CFU-GM can obtain from peripheral blood, bone marrow, tumor-infiltrated cell, tumor surrounding tissue infiltrating cells, lymph node, spleen, skin, Cord blood or any other suitable tissue or body fluid.For example, can, by the combination of cytokine for example GM-CSF, IL-4, IL-13 and/or TNF α being added to from the monocyte cultures of peripheral blood results, make dendritic cell ex vivo differentiation.Or, can, by the compound of GM-CSF, IL-3, TNF α, CD40L, LPS, flt3 part and/or other induction dendritic cell differentiation, maturation and propagation is joined in described culture medium, make to be divided into dendritic cell from the CD34 positive cell of peripheral blood, Cord blood or bone marrow results.

Dendritic cell routine is divided into " immaturity " cell and " maturation " cell, and available simple mode makes a distinction two kinds of phenotypes that fully characterize.But this name should not be interpreted as getting rid of all possible middle differential period.The antigen relevant with mannose receptor high expressed to Fc γ receptor that is characterized as of immaturity dendritic cell is taken in and the high APC of working ability.Ripe phenotype is common is characterised in that the expression of these labellings is lower, but it is high to be responsible for the expression of cell surface molecule (for example I class MHC and II class MHC), adhesion molecule (for example CD54 and CD11) and co stimulatory molecule (for example CD40, CD80, CD86 and 4-1BB) of T cell activation.

Generally can, with polynucleotide of the present invention (or its part or other variant) transfection APC, make coded polypeptide or its immunogenicity part at described cell surface expression.Can carry out such transfection in vitro, the Pharmaceutical composition that then comprises such transfectional cell can be for therapeutic purposes as herein described.Or, can give patient by the gene delivery vector of targeting dendritic cells or other antigen-presenting cell, cause occurring in vivo transfection.Generally speaking, adopt any method known in the art, the method or the Mahvi etc. that such as in WO 97/24447, introduce, Immunology and cell Biology 75:456-460, the 1997 particle gun methods of introducing, can carry out in the body of for example dendritic cell and in vitro transfection.By hatching together with dendritic cell or CFU-GM and oncopeptide, DNA (naked DNA or at the DNA in plasmid vector) or RNA; Or for example, hatch together with the recombinant bacteria of antigen expressed or virus (vaccinia virus, fowlpox virus, adenovirus or lentivirus carrier), the antigen that can reach dendritic cell loads.Before loading, immune spouse auxiliary with T cell is provided polypeptide can be stopped to (for example carrier molecule) covalency and put together.Or, dendritic cell can be individually or under described polypeptide exists by unconjugated immune gametophyte burst process.

The in the situation that of can using any suitable carrier well known by persons skilled in the art in Pharmaceutical composition of the present invention, bearer type becomes with mode of administration conventionally.Compositions of the present invention can be prepared for any suitable administering mode, comprises for example local, oral, nasal cavity, mucosa, intravenous, intracranial, intraperitoneal, subcutaneous and intramuscular administration.

Biocompatible for the carrier of this class Pharmaceutical composition, and also biodegradable.In certain embodiments, described preparation preferably provides with relative constant level and discharges effective ingredient.But, in other embodiments, may after administration, discharge immediately effective ingredient with the speed of more accelerating.Adopting known technology to prepare this based composition is in the knowledge of those skilled in the range.The illustrative carrier that can be used for this aspect comprises microgranule of PLGA, polyacrylate, latex, starch, cellulose, glucosan etc.The carrier of other illustrative delayed release comprises supermolecule bio-carrier, it comprises immobilising hydrophilic core (for example cross-linked polysaccharides or oligosaccharide) and the optional skin that comprises amphipathic compound (for example phospholipid) (referring to for example, United States Patent (USP) the 5th, 151, No. 254 and PCT application WO 94/20078, WO 94/23701 and WO 96/06638).In slow releasing preparation, the amount of contained reactive compound depends on speed and expected duration and the disease character to be treated or prevention of implant site, release.

In another illustrative embodiment, biodegradable microsphere (for example poly-lactic acid ester PVOH acid esters) is as the carrier of the present composition.Suitable biodegradable microsphere is disclosed in for example U.S. Patent number 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344,5,407,609 and 5,942,252.Modified hepatitis B virus carrier system, the carrier system of for example, describing in WO/99 40934 and the list of references wherein quoted, also can be used in many application.Another illustrative carrier/transmission system is used the carrier that comprises graininess albumen composition, for example, at United States Patent (USP) the 5th, and the carrier of describing in 928, No. 647, described carrier can induce the cytotoxic T lymphocyte of I class restriction to reply in host.

Pharmaceutical composition of the present invention also comprises one or more buffer agents (for example neutral buffered saline solution or phosphate buffered saline(PBS)) conventionally, saccharide (for example glucose, mannose, sucrose or glucosan), mannitol, protein, polypeptide or aminoacid (for example glycine), antioxidant, antibacterial, chelating agen (for example EDTA) or glutathion, adjuvant (for example aluminium hydroxide), blood that makes preparation and receptor etc. oozes, the solute that hypotonic or weak height oozes, suspending agent, thickening agent and/or antiseptic.Or the present composition can be formulated as lyophilized formulations.

Pharmaceutical composition as herein described for example can be contained in, in unit-dose container or multi-dose container (sealed ampoule or phial).The common sealing means of this class container makes to keep the aseptic and stability of described preparation before using.Generally speaking suspensoid, solution or Emulsion that, preparation can be used as in oiliness or aqueous vehicles are preserved.Or Pharmaceutical composition can be preserved under freeze-dried condition, only sterile liquid carrier need to added before use.

For use particular composition as herein described in various therapeutic schemes for, the exploitation of suitable dosage regimen and therapeutic scheme (comprising for example oral, parenteral, intravenous, intranasal and intramuscular administration and preparation used) is well-known in the art, in order to illustrate, below some scheme wherein will be discussed in a capsule.

In some applications, Pharmaceutical composition disclosed herein can be passed to animal by oral administration.Therefore, these compositionss can be prepared with inert diluent or together with absorbable edible carrier, or they can be encapsulated in duricrust gelatine capsule or soft shell gelatin capsules, or they can be squeezed into tablet, or they can directly be incorporated in the food of meals.

Described reactive compound even may mix together with excipient, with edible tablet, buccal tablet, lozenge, capsule, elixir, suspensoid, syrup, wafer form etc. use (referring to, for example, Mathiowitz etc., Nature on March 27th, 1997; 386 (6623): 410-4; Hwang etc., Crit Rev Ther Drug Carrier Syst 1998; 15 (3): 243-84; United States Patent (USP) 5,641,515; United States Patent (USP) 5,580,579 and United States Patent (USP) 5,792,451).Tablet, lozenge, pill, capsule etc. also can contain any in many additional component, can add for example binding agent, for example tragakanta, Radix Acaciae senegalis, corn starch or gelatin; Excipient, for example dicalcium phosphate; Disintegrating agent, such as corn starch, potato starch, alginic acid etc.; Lubricant, for example magnesium stearate; And sweeting agent, for example sucrose, lactose or glucide or correctives, for example Herba Menthae, wintergreen oil or Fructus Pruni pseudocerasi correctives.In the time that dosage unit form is capsule, except the above-mentioned type material, it can also contain liquid-carrier.Various other materials may exist as coating materials, or improve the physical form of dosage unit in other side.For example, tablet, pill or capsule can or be used Lac and sweet tablet with Lac, sugar simultaneously.Certainly, preparation any dosage unit form in any material used should be all pharmacy pure and in used amount, be nontoxic substantially.In addition, described reactive compound can be incorporated in slow releasing preparation.

Conventionally, these preparations contain at least about 0.1% reactive compound or the reactive compound of high dose more, although the percentage rate of effective ingredient can change certainly, may conventionally be total weight of formulation or body knit approximately 1% or 2% to approximately 60% or 70% or higher.Certainly, in every kind that may prepare is treated effective compositions, the mode of the consumption of reactive compound makes to obtain suitable dosage with any given unit dose of described compound.In the time of this pharmaceutical formulation of preparation, those skilled in the art can consider that therefore various dosage and therapeutic scheme may be all desirable such as dissolubility, bioavailability, biological halflife, route of administration, the factors of goods storage period and other pharmacology consideration.

On the other hand, for oral administration, compositions of the present invention can be mixed one or more excipient in mouth-wash, dentifrice, buccal tablet, oral spray or Sublingual oral Preparation form.Or, described effective ingredient can be incorporated in the oral administration solution that oral administration solution for example contains sodium borate, glycerol and sodium bicarbonate, or in dentifrice, disperse, or join in the compositions that may comprise water, binding agent, abrasive, correctives, foaming agent and wetting agent with treatment effective dose.Or described compositions can be finish-machined to can put into Sublingual or tablet or solution form at dissolved in oral cavity.

In some cases, it is desirable to parenteral, intravenous, intramuscular or even intraperitoneal give Pharmaceutical composition disclosed herein.Such method is well known to those skilled in the art, and wherein some method is further described in for example United States Patent (USP) 5,543,158; United States Patent (USP) 5,641,515 and United States Patent (USP) 5,399,363.In certain embodiments, in water, suitably for example, mix with surfactant (hydroxypropyl cellulose), can prepare the solution of described reactive compound as free alkali or the upper acceptable salt of pharmacology.Also can prepare dispersant at glycerol, liquid macrogol and composition thereof with in oil.Under common storage and service condition, these preparations generally contain antiseptic, to prevent microbial growth.

The illustrative medicine that is suitable for injection forms and comprises aseptic aqueous solution or dispersant and for the aseptic injection of interim preparation or the sterilized powder (for example,, referring to United States Patent (USP) 5,466,468) of dispersion liquid.In all cases,, described form must be aseptic and must be liquid being easy to aspect injection.Produce and the condition of storage under it must be stable and must prevent the contamination of microorganism (for example antibacterial and fungus).Described carrier can be solvent or contain such as water, ethanol, polyhydric alcohol (such as glycerol, propylene glycol and liquid macrogol etc.), their suitable mixture and/or the disperse medium of vegetable oil.For example, by utilizing the coating materials such as lecithin, in the situation that disperseing, by keeping required granular size and/or utilizing surfactant, can keep suitable mobility.Can utilize various antibacterial agents or antifungal agent (for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal etc.) to prevent the effect of microorganism.In many cases, preferably include isotonic agent, for example sugar or sodium chloride.The prolongation of injectable composition absorbs can be by using the medicament that postpones absorption in compositions, and for example aluminum monostearate and gelatin are realized.

In one embodiment, give aqueous solution for parenteral, described solution should suitably cushion if desired, described liquid diluent first with enough saline or glucose make its etc. ooze.These specific water solublity are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.At this on the one hand, according to disclosure of the present invention, operable sterile aqueous media is known for those skilled in the art.For example, a dosage can be dissolved in 1ml isotonic sodium chlorrde solution, then or join 1000ml subcutaneous perfusion liquid or in the infusion site injection of suggestion (referring to for example, " Remington ' s Pharmaceutical Sciences ", the 15th edition, 1035-1038 page and 1570-1580 page).According to curee's to be treated disease, some variation of dosage is essential.In addition,, for mankind's administration, certainly preparation preferably meets aseptic, apyrogeneity and according to the desired general safety of FDA Office of Biologics standard and purity rubric.

In another embodiment of the invention, compositions disclosed herein can be mixed with neutral form or salt form.Illustrative pharmaceutically acceptable salt comprises acid-addition salts (generating with the free amine group of protein), and the acid-addition salts that generates with the mineral acid of all example hydrochloric acids or phosphoric acid or with the organic acid of such as acetic acid, oxalic acid, tartaric acid, mandelic acid etc.With free carboxy form salt also can be derived from inorganic base, for example sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or hydrated ferric oxide.; And organic base, such as 2-aminopropane., triethylamine, histidine, procaine etc.In the time of preparation, the mode that solution gives need be compatible with drug-delivery preparation, and the dosage giving is treatment effective dose.

Described carrier can also comprise any and all solvents, disperse medium, solvent, coating materials, diluent, antibacterial agent and antifungal agent, isotonic agent and absorption delay agent, buffer agent, carrier solution, suspensoid, colloid etc.This class medium and medicament that hyoscine active substance uses are well-known in the art.Except any conventional media and medicament and the inconsistent situation of effective ingredient, consider its application in therapeutic combination.Also supplementary effective ingredient can be incorporated in described compositions.Phrase " pharmaceutically acceptable " refers to the molecular entity and the compositions that in the time giving the mankind, do not produce allergy or similar undesired reaction.

In certain embodiments, described Pharmaceutical composition can spray by intranasal, suck and/or other aerosol transmits carrier and transmits.The method that directly gives pulmonary by nose aerosol spray by gene, nucleic acid and peptide combinations is described in for example United States Patent (USP) 5,756,353 and United States Patent (USP) 5,804,212.Equally, adopt intranasal finely divided resin (Takenaga etc., J Controlled Release1998 March 2; 52 (1-2): 81-7) and lysophosphatidyl glycerol compound (United States Patent (USP) 5,725,871) administration be also that pharmaceutical field is well-known.Equally, the illustrative mucosal of politef support skeleton form is at United States Patent (USP) 5,780, has description in 045.

In some embodiment, use liposome, nanocapsule, microgranule, lipid granule, vesicle etc. that compositions of the present invention is introduced in suitable host cell/biology.Specifically, compositions of the present invention can be mixed with in encapsulation transmission such as lipid granule, liposome, vesicle, nanosphere or nanoparticles.Or, compositions of the present invention can with such carrier surface or covalent bond or non-covalent combination.

Liposome and liposome sample preparation as the preparation of potential drug carrier and application be generally for those skilled in the art known (referring to for example, Lasic, Trends Biotechnol in July, 1998; 16 (7): 307-21; Takakura, Nippon Rinsho in March, 1998; 56 (3): 691-5; Chandran etc., Indian J Exp Biol.1997 August; 35 (8): 801-9; Margalit, CritRev Ther Drug Carrier Syst.1995; 12 (2-3): 233-61; United States Patent (USP) 5,567,434; United States Patent (USP) 5,552,157; United States Patent (USP) 5,565,213; United States Patent (USP) 5,738,868 and United States Patent (USP) 5,795,587; Each document is all attached to herein particularly by reference).

Liposome successfully uses together with the cell type of other method transfection with many being conventionally difficult to, and comprises T cell suspensoid, primary hepatocyte culture and PC 12 cells (Renneisen etc., J Biol Chem.1990 JIUYUE 25 days; 265 (27) .16337-42; Muller etc., DNA Cell Biol.1990 April; 9 (3): 221-9).In addition, liposome is not based on common DNA length restriction in viral drug-supplying system.Liposome is used for gene, various medicine, radiotherapy medicine, enzyme, virus, transcription factor, allosteric effect agent etc. to import in various cultured cell systems and animal effectively.In addition, it seems liposome application be administered systemically after autoimmunity reply or unacceptable toxicity irrelevant.

In certain embodiments, liposome is formed and is automatically formed the double-deck vesicle of multilayer concentric (also referred to as multilamellar liposome (MLV)) by the phospholipid disperseing in aqueous medium.

On the other hand, in other embodiments, the invention provides the pharmaceutically acceptable nanocapsule preparation of the present composition.Nanocapsule generally can with stablize with reproducible mode encapsulation compound (referring to, for example, Quintanar-Guerrero etc., Drug Dev Ind Pharm.1998 December; 24 (12): 1113-28).For fear of the side effect that overloads and cause due to intracellular polymer, (size approximately 0.1 μ m can adopt polymer that can vivo degradation to design for such ultramicro powder.Such granule can be prepared described in following document: such as Couvreur etc., Crit Rev Ther Drug Carrier Syst.1988; 5 (1): 1-20; Zur Muhlen etc., Eur JPharm Biopharm.1998 March; 45 (2): 149-55; Zambaux etc., J ControlledRelease.1998 January 2; 50 (1-3): 31-40; With United States Patent (USP) 5,145,684.

The present invention also relates to the polynucleotide of the primer form that derives from polynucleotide of the present invention and specificity for the antibody of polypeptide of the present invention or the polypeptide of reagent form the purposes as diagnostic reagent.

Evaluation can detect utmost point early changes in carcinogenesis approach blood or tissue in genetic marker or biochemical marker, will contribute to be identified for patient's best therapy.Can use the alternative tumor marker of expressing such as polynucleotide, diagnose multi-form and cancer stadium.The evaluation of polynucleotide expression of the present invention both be can be used for to the stadium classification of Cancerous disease, can be used for again the character classification of cancerous tissue.The progress of stadium sorting technique monitoring cancer, and according to whether existing malignant tumor tissue to determine in biopsy district.Polynucleotide of the present invention can contribute to be tested and appraised the labelling of cancer attack, described stadium sorting technique is improved in for example existence in body different parts.How closely similar the hierarchical description tumor of cancer is to the normal structure of its same type, and evaluate by its cellular morphology and other differentiation marker.Polynucleotide of the present invention can be used for determining tumor rank, because they may contribute to determine the differentiation state of tumor cell.

By comprise determine derive from the abnormal minimizing of polypeptide in curee's sample or mRNA level or be increased in method diagnose, it is a kind of for cancer diagnosis, autoimmune disease and associated conditions or determine the method for the susceptibility to described disease that described diagnostic analysis provides.This diagnostic method is called differential expression.The relatively expression of specific gene in illing tissue and normal structure.Difference described in these two kinds of tissues that compared between polynucleotide related gene, mRNA or albumen, the for example difference aspect molecular weight, aminoacid sequence or nucleotide sequence or relative abundance, shows suspecting gene described in ill people's tissue or regulating the variation of its gene.

Can on rna level, measure minimizing or the increase expressed.First from these two kinds of tissues, isolate PolyA RNA, then can be detected by the following method by the detection of the mRNA of the gene code of the polynucleotide of the present invention corresponding to differential expression: in situ hybridization, the reverse transcription-PCR of for example tissue slice, use RNA trace or any other direct or indirect RNA detection method containing PolyA+mRNA.The increase that given RNA expresses in illing tissue compared with normal structure or minimizing, point out described transcript and/or expressed albumen to work in described disease.Therefore, detect that to correspond to the mRNA level of SEQ ID NO:1 more high or low than normal level, show that described patient suffers from cancer.

Mrna expression level in sample can be determined by the library that produces expressed sequence tag (EST) from described sample.Can use the relative expressivity of EST in described library, evaluate the relative expressivity of described genetic transcription thing in primary sample.Then the EST of described test can be analyzed with the EST of reference sample and analyzes and compare, thereby measure relative expression's level of interested polynucleotide.

Can adopt continuous analysis (SAGE) method (Velculescu etc. of gene expression, Science (1995) 270:484), mRNA differential display mRNA method (for example, US 5,776,683) or depend on the nucleotide narrow spectrum hybridization analysis that interacts, carry out other mRNA analysis.

On the other hand, can on protein level, compare.Use antibody test from the polypeptide in the Western blotting of the protein extract in described two kinds of tissues, can compare the protein size in two kinds of tissues.Use the antibody of anti-corresponding protein, can also in immunology, detect expression and Subcellular Localization.Can be used for measuring other analytical technology of the protein level (for example polypeptide level of the present invention) in host-derived sample, know to those skilled in the art.In illing tissue, expression of polypeptides level raises or reduces compared with same protein expression in normal structure, shows that expressed albumen may be relevant with described disease.

In mensuration of the present invention, by detecting the expression by the gene outcome of at least one sequential coding shown in SEQ ID NO:1, can determine described diagnosis.Also can, with the mRNA level in illing tissue and normal structure or the comparison of protein level, follow the tracks of advancing of disease or alleviation.

Adopt polynucleotide arrays, the many polynucleotide sequences in can analytic sample.These polynucleotide can be used for detecting the differential expression of gene, thereby determine the function of gene.For example, can determine with the array of polynucleotide sequence SEQ ID NO:1 any whether variant expression between normal cell and cancer cell of described polynucleotide.In one embodiment of the invention, can build the array of an oligonucleotide probe that comprises SEQ ID NO:1 nucleotide sequence or its fragment, to carry out the Effective selection of for example genetic mutation.Array technique method is well-known and has general applicability, can be by the various problems that solve molecular genetics aspect, comprise that gene expression, genetic linkage and hereditary variability are (referring to for example: M.Chee etc., Science, the 274th volume, 610-613 page (1996)).

" diagnosis " used herein comprises and determines the susceptibility of curee to disease, determines that whether curee suffers from described disease at present, can also do prognosis to the curee who is subject to described sickness influence.

The invention still further relates to the diagnostic kit for carrying out diagnostic analysis, described test kit comprises:

(a) polynucleotide of the present invention or its fragment, described polynucleotide are the nucleotide sequence of SEQID NO:1 preferably;

(b) nucleotide sequence of one and nucleotide sequence complementation (a), the preferably nucleotide sequence of SEQ IDNO:6;

(c) polypeptide of the present invention or its fragment, described polypeptide is the polypeptide of SEQ ID NO:2 or SEQ ID NO:3 preferably; Or

(d) antibody for anti-polypeptide of the present invention, the preferably antibody of anti-SEQ ID NO:2 or SEQ IDNO:3 polypeptide.

For chromosome mapping, nucleotide sequence of the present invention is also valuable.Ad-hoc location on described sequence-specific ground targeted human individual chromosome, and can be hybrid with it.According to the present invention, relevant sequence being mapped to chromosome, is the important first step making those sequences and gene-correlation disease in contacting.Once the exact position that sequence is mapped to chromosome, just can make the physical location of described sequence on described chromosome be associated with gene mapping data.Such data can be at for example V.McKusick, in Mendelian Inheritance inMan (can obtain online by Johns Hopkins University Welch Medical Library), finds.Then identify the relation of having mapped between gene and the disease of same chromosomal region by linkage analysis (the physically common heredity of adjacent gene).Also can determine the difference in cDNA sequence or the genome sequence between affected individuals and uninfluenced individuality.

Polypeptide of the present invention or its fragment or its analog or the cell of expressing them can also be used as immunogen, to produce the immune specificity antibody of anti-polypeptide of the present invention.Term " immune specificity " refers to that described antibody is more much higher to the affinity of other related polypeptide in prior art than them to the affinity of polypeptide of the present invention.

On the one hand, the invention provides as defined above according to the immune specificity antibody of polypeptide of the present invention or its immunology fragment again.Preferably monoclonal antibody of described antibody.

Adopt conventional method, by described polypeptide or the fragment, analog or the cell that carry epi-position being given to animal, non-human animal preferably, can obtain the antibody producing for polypeptide of the present invention.In order to prepare monoclonal antibody, can use any technology that the antibody of being produced by continuous cell line culture is provided.Example comprises hybridoma technology (Kohler, and Milstein G., C., Nature (1975) 256:495-497), trioma technology-human B-lymphocyte hybridoma technology (Kozbor etc., Immunology Today (1983) 4:72) and EBV hybridoma technology (Cole etc., MonoclonalAntibodies and Cancer Therapy, 77-96, Alan R.Liss, Inc., 1985).

Produce the technology of single-chain antibody, for example, at United States Patent (USP) the 4th, the technology of describing in 946, No. 778, may also be applicable to produce the single-chain antibody of anti-polypeptide of the present invention.In addition, can express humanized antibody with transgenic mice or other biology (comprising other mammal).

Can use above-mentioned antibody to separate or identify and express the clone of described polypeptide or by polypeptide described in affinitive layer purification.Also can use antibody prevention of the present invention or treatment cancer, especially colorectal carcinoma, autoimmune disease and associated conditions.

Another aspect of the present invention relates to induction or regulates the method for immunne response in mammalian body; described method comprises with being enough to produce mammal described in the peptide vaccination of the present invention of antibody and/or T cellullar immunologic response, to protect or to improve symptom or the process of described disease.The method that relates in one aspect to again induction or regulate immunne response in mammalian body of the present invention; described method comprises the carrier transfer polypeptide of the present invention of expressing described polynucleotide coding said polypeptide by instructing in vivo; so that induction produces this immunne response of antibody, thereby protects described animal to exempt from the disease that takes a disease.

Therefore people will appreciate that, the invention provides treatment and the not enough relevant for example cancer of abnormal disease of existence, overexpression or expression of CASB7439 polypeptide active and the method for autoimmune disease, especially colorectal carcinoma.The present invention attempts other for the treatment of, and to express relevant abnormal disease with CASB7439 be chronic lymphocytic leukemia and germ cell tumor.

The present invention also provides SCREENED COMPOUND to identify the method that stimulates or suppress those compounds of CASB7439 polypeptide function.Generally speaking, for this class disease mentioned above, in order to treat and to prevent object can use agonist or antagonist.Can carry out authenticating compound by for example mixture of cell, acellular prepared product, chemical library and natural product from various sources.As the case may be, this excitomotor, antagonist or the inhibitor of so identifying can be the natural or modified substrate, part, receptor, enzyme of described polypeptide etc., or can be also their structural simulation thing or functional simulation thing (referring to Coligan etc., Current Protocols inImmunology the 1 (2): the 5th chapter (1991)).Screening technique is well known by persons skilled in the art.Other screening technique can also be at such as D.Bennett etc., J Mol Recognition, 8:52-58 (1995); With K.Johanson etc., J Mol Biol, 270 (16): find in 9459-9471 (1995) and list of references wherein.

Therefore, the invention provides and identify the screening technique that stimulates or suppress the compound of polypeptide function of the present invention, described method comprises a kind of following method that is selected from:

(a) utilize the directly or indirectly labelling in conjunction with candidate compound, measure described candidate compound and described polypeptide (or with the cell or the cell membrane that carry described polypeptide) or the combination with its fusion rotein;

(b), under the existence of labelling competition thing, measure candidate compound and described polypeptide (or with the cell or the cell membrane that carry described polypeptide) or the combination with its fusion rotein;

(c) use the detection system that is suitable for the cell or the cell membrane that carry described polypeptide, test described candidate compound and whether cause the signal producing due to activation or the inhibition of described polypeptide;

(d) candidate compound is mixed with the solution containing claim 1 polypeptide, form mixture, then measure the activity of polypeptide described in described mixture, and the activity of the activity of described mixture and standard substance is compared; Or

(e) for example adopt ELISA to measure, detect the impact of the generation of the mRNA of candidate compound on coding said polypeptide in cell and described polypeptide.

By standard receptors bind technology known in the art, polypeptide of the present invention also can be used to identify membrane-bound receptor or soluble recepter (if any).Can also identify and agonist and the antagonist (if any) of polypeptide competition of the present invention with the polypeptide of the present invention of its receptors bind with well-known screening technique.

Therefore, on the other hand, the present invention relates to for the identification of the agonist of polypeptide of the present invention, antagonist, part, receptor, substrate, enzyme etc.; Or the screening reagent box that reduces or increase the compound of this class polypeptide generation, described test kit comprises:

(a) a kind of polypeptide of the present invention;

(b) a kind of reconstitution cell of expressing polypeptide of the present invention;

(c) a kind of cell membrane of expressing polypeptide of the present invention; Or

(d) antibody of anti-polypeptide of the present invention;

Described polypeptide is the polypeptide of SEQ ID NO:2 or SEQ ID NO:3 preferably.

Those skilled in the art can easily recognize, polypeptide of the present invention can also be used for the method for agonist, antagonist or inhibitor based on polypeptide described in structural design, and using method comprises:

(a) three dimensional structure of first definite described polypeptide;

(b) the possible active site of derivation agonist, antagonist or inhibitor or the three dimensional structure of binding site;

(c) candidate compound that synthetic prediction is combined or is reacted with binding site or the active site of derivation; With

(d) whether test described candidate compound is real agonist, antagonist or inhibitor.

Can also use gene therapy to realize by the cells involved endogenous in curee's body and produce CASB7439 polypeptide.The summary of related gene treatment, referring to Human Molecular Genetics, the 20th chapter, gene therapy and other Therapeutic Method (Gene Therapyand other Molecular Genetic-based Therapeutic Approaches) based on molecular genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996) (with the list of references of wherein quoting).

At Pharmaceutical Biotechnology, the 61st volume, vaccine design-subunit and adjuvant method (Vaccine Design-the subunit and adjuvant approach), Powell and Newman write, Plenurn Press, 1995.New Trends and Developments inVaccines, Voller etc. write, University Park Press, Baltimore, Maryland has described bacterin preparation comprehensively in U.S.A.1978.With the liposomal encapsulated United States Patent (USP) 4,235,877 that is described in for example Fullerton.Protein and macromole are puted together the United States Patent (USP) 4,474,757 of the United States Patent (USP) 4,372,945 and the Armor etc. that are disclosed in for example Likhite.

Albumen quality in every kind of vaccine dose is chosen to be induction of immunity protective response in typical vaccine and without the amount of obvious toxic-side effects.Such amount changes with used specific immunogen.Generally speaking, expect that each dosage comprises 1-1000 μ g albumen, preferably 2-100 μ g albumen, 4-40 μ g albumen most preferably.By comprising that observation curee internal antibody is tired and the research on standard of other reaction, can determine the optimised quantity of specific vaccine.After first vaccination, curee can also accept a booster shot in about 4 weeks.

" separation " refer to from native state " artificially " change.If a kind of " separation " compositions or material exist at occurring in nature, its primal environment has changed or from its primal environment, has taken out or both have both at the same time so.As this term used herein, for example in the animal living, naturally occurring polynucleotide or polypeptide are not " separation ", but described identical polynucleotide or polypeptide that the material coexisting with its native state separates are " separation ".

" polynucleotide " generally refer to any polyribonucleotide or polydeoxyribonucleotide, and they can be RNA or DNA or modified RNA or the DNA that comprises the unmodified of strand district and double stranded region.

" variant " refers to different from reference polynucleotide or polypeptide but retains polynucleotide or the polypeptide of fundamental characteristics.Typical case's polynucleotide variant lists different from another reference polynucleotide at nucleotides sequence.The variation of described variant nucleotide sequence may change or not change the aminoacid sequence of the polypeptide of reference polynucleotide encoding.As discussed below, the change of nucleotide may cause propylhomoserin replacement, interpolation, disappearance, fusion and the truncate of the polypeptide of reference sequence coding.Typical polypeptide variants is different from another reference polypeptide on aminoacid sequence.Generally speaking, difference is limited, and the sequence that causes reference polypeptide and variant is closely similar and be identical in many districts on the whole.Variant and reference polypeptide may be due to one or more replacements of any combination, interpolation, disappearance and aminoacid sequence is different.The amino acid residue that the amino acid residue replacing or insert can yes or no be encoded by genetic code.The variant of polynucleotide or polypeptide can be naturally occurring variant as allelic variation body, or described variant can be the variant that known non-natural exists.Can, by induced-mutation technique or by directly synthetic, prepare the variant of the non-natural existence of polynucleotide and polypeptide.

" homogeneity ", as known in the art, refers to the relation between two or more peptide sequence or two or more polynucleotide sequence definite by comparative sequences.In the art, as the case may be, " homogeneity " also refers to according to the coupling between the character string of polypeptide or polynucleotide sequence and serial correlation degree between definite polypeptide or polynucleotide sequence.Adopt known method can easily calculate " homogeneity " and " similarity ", described method includes but not limited to the method to describe in Publication about Document: Computational MolecularBiology, Lesk, A.M. write, Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D.W. writes, Academic Press, New York, 1993; Computer Analysis of Sequence Data, part i, Griffin, A.M. and Griffin, H.G. writes, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; With Sequence Analysis Primer, Gribskov, M. and Devereux, J. writes, M Stockton Press, New York, 1991; And Carillo, H. and Lipman, D., SIAM J.Applied Math., 48:1073 (1988).The method for optimizing of design mensuration homogeneity is the maximum match between cycle tests in order to provide.The method of homogeneity and similarity of measuring has been compiled into the available computer program of the public.Include but not limited to GCG program package (Devereux for measuring two homogeneity between sequence and the preferred computer program technic of similarity, J. etc., 387 (1984)), BLASTP, BLASTN and FASTA (Atschul Nucleic Acids Research 12 (1):, S.F. etc., J.Molec.Biol.215:403-410 (1990)).The public can obtain BLAST X program (BLAST Manual, Altschul, S. etc., NCBINLM NIH Bethesda, MD 20894 from NCBI and other source; Atschul, S. etc., J.Mol.Biol.215:403-410 (1990)).Also can use well-known Smith Waterman algorithm to measure homogeneity.

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

Gap Penalty (gap penalty): 12

Gap extension penalty (room expansion point penalty): 4

Word size (word string size): 2, be 6 to the maximum

Comprise following parameter with the preferred parameter of the many peptide sequences of other method:

1) algorithm: Needleman and Wunsch, J.Mol Biol.48:443-453 (1970)

Comparison matrix (comparator matrix): the BLOSSUM62 of Hentikoff and Hentikoff, Proc.Natl.Acad.Sci.USA.89:10915-10919 (1992)

Gap Penalty：12

Gap Length penalty (room length point penalty): 4

The program that can use these parameters is " gap " program, and the public can be from Genetics ComputerGroup, and Madison WI obtains.The parameter mentioned is above the default parameter (simultaneously end room not being had to point penalty) that carries out polypeptide comparison.

Preferred parameter for polynucleotide comparison comprises following parameter:

1) algorithm: Needleman and Wunsch, J.Mol Biol.48:443-453 (1970)

Comparison matrix:matches (coupling)=+ 10, mismatch (mispairing)=0

Gap Penalty：50

Gap Length penalty：3

The program that can use these parameters is " gap " program, and the public can be from Genetics ComputerGroup, and Madison WI obtains.The parameter mentioned is above the default parameter that carries out polynucleotide comparison.

As an example, polynucleotide sequence of the present invention can be identical with the reference sequence of SEQ ID NO:1, and namely 100% is identical, or it can comprise compared with described reference sequence, and the nucleotide of a certain integer of as many as changes.Such change is selected from least one nucleotide deletion, replacement (comprising base transition and transversion) or inserts, and wherein said change can occur between 5 ' or 3 ' terminal position of described contrast nucleotide sequence or this two terminal positions Anywhere, or intersperse among separately separately in the nucleotide of reference sequence, or intersperse among in one or more continuous group of reference sequence.The number that nucleotide changes is determined as follows: SEQ ID NO:1 nucleotide sum is multiplied by the percentile numerical value percentage rate of corresponding homogeneity (divided by 100), deducts this product subsequently from described SEQ ID NO:1 nucleotide sum, or

n _n≤x _n-(x _n·y)，

Wherein n _nthe number that nucleotide changes, x _nbe SEQ ID NO:1 nucleotide sum, y is for example 0.70 (70%), 0.80 (80%), 0.85 (85%), 0.90 (90%), 0.95 (95%) etc., and wherein by x _nwith any non-integer product of y from x _nbefore deducting, this product is rounded to nearest integer.The change of the polynucleotide sequence of coding SEQ ID NO:2 polypeptide may produce nonsense, missense or frameshift mutation in this coded sequence, thereby changes afterwards the polypeptide by described polynucleotide encoding in such change.

Equally, peptide sequence of the present invention can be identical with the reference sequence of SEQ ID NO:2, and namely 100% is identical, or it can comprise compared with described reference sequence, the amino acid change of a certain integer of as many as, so that homogeneity percentage rate is less than 100%.Such change is selected from least one aminoacid deletion, replacement (comprising conservative replacement and non-conservative replacement) or inserts, and wherein said change can occur between the amino terminal of described reference peptide sequence or carboxyl terminal position or this two terminal positions Anywhere, or intersperse among separately separately in the aminoacid of reference sequence, or intersperse among in one or more continuous group of reference sequence.Determine as follows the number of described amino acid change for specific homogeneity percentage rate: SEQ ID NO:2 aminoacid sum is multiplied by the percentile numerical value percentage rate of corresponding homogeneity (divided by 100), deduct this product from described SEQ IDNO:2 aminoacid sum subsequently, or

n _a≤x _a-(x _a·y)，

Wherein n _athe number of amino acid change, x _abe SEQ ID NO:2 aminoacid sum, y is for example 0.70 (70%), 0.80 (80%), 0.85 (85%) etc., and wherein by x _awith any non-integer product of y from x _abefore deducting, this product is rounded to nearest integer.

" homologue " is the term of this area classification used, refers to the polynucleotide sequence or the peptide sequence that have with subject nucleotide sequence height serial correlation.Such dependency can be by determining homogeneity degree between institute as above comparative sequences and/or similarity degree quantitatively.Belong within the scope of this classification term is term " straight homologues " and symbiosis congener "; " straight homologues " refers to polynucleotide or the polypeptide of the function equivalent in another species as a kind of polynucleotide or polypeptide, and " symbiosis congener " refers to and in same species, be considered to sequence similar in function.

Accompanying drawing explanation

fig. 1: show the PCR in real time data that adopt Taqman probe.Described being described as follows: adrenal gland: Ad_G1; Bladder: B1; Bone marrow: Bo_Ma; Cervix uteri: Ce; Colon: Co; Fallopian tube: Fa_Tu; Ileum: I1; Liver: Li; Lung: Lu; Lymph node: Ly_No; Esophagus: Oe; Parathyroid gland: Pa_Thy; Placenta Hominis: P1; Prostate: Pr; Rectum: Re; Skin: Sk; Skeletal muscle: Sk_Mu; Small intestinal: Sm_In; Spleen: Sp; Testis: Te; Thyroid: Thy; Trachea: Tr.

fig. 2show and adopt the PCR in real time of Sybr scheme to express.Described being described as follows: adrenal gland: Ad_G1; Bladder: B1; Bone marrow: Bo_Ma; Cervix uteri: Ce; Colon: Co; Lymph node: Ly_No; Esophagus: Oe; Parathyroid gland: Pa_Thy; Placenta Hominis: P1; Prostate: Pr; Rectum: Re; Skin: Sk; Skeletal muscle: Sk_Mu; Small intestinal: Sm_In; Spleen: Sp; Testis: Te; Thyroid: Thy; Trachea: Tr; Heart: He.

fig. 3show the SDS PAGE of the Coomassie blue stain of the cell extract that derives from the bacterial strain of expressing CASB7439.Swimming lane 1 shows molecular weight marker, and swimming lane 2 is shown in 39 ℃ of inductions cell extract of 5 hours; Swimming lane 3 shows the supernatant of induced cell extract; Swimming lane 4 shows the precipitation of induced cell extract.

fig. 4show the western blot analysis of NS1-CASB7439 expressing protein.By the cell extract application of sample of bacterial strain of expressing CASB7439 to this gel, and with anti-NS1 monoclonal antibody announcement.

fig. 5show the SDS-PAGE of the Coomassie blue stain of CASB7439 after purification.Swimming lane 1 and swimming lane 5 represent molecular weight marker; Swimming lane 2,3,4 adds respectively 2 μ l, 4 μ l and 6 μ l purifying proteins.

fig. 6show the Western blotting of the CASB7439 disclosing according to anti-poly histidine monoclonal antibody after purification.

embodiment

embodiment 1

Real-time RT-PCR is analyzed

Use real-time RT-PCR (U.Gibson.1996.Genome Research:6,996) compares the abundance from the mRNA transcript of candidate antigens in the tumor colon of multiple patients' pairing and normal colonic tissue.In addition, evaluate the mRNA level of candidate gene described in one group of normal structure by the method.

Use TriPure reagent (Boehringer), from quick-freezing biopsy, extract total RNA of Normal Colon and tumor colon.Total RNA of normal structure is purchased from In Vitrogen or use TriPure reagent to extract from quick-freezing biopsy.Use oligodeoxythymidylic acid magnetic bead (Dynal), from purification Poly-A through DNA enzyme total RNA after treatment ⁺mRNA.Use SybrII dyestuff (Molecular Probes), carry out the quantitative of mRNA by spectrofluorimetry (VersaFluor, BioRad).Adopt the default option of TaqMan amplification condition, the primer by Perkin-ElmerPrimer Express software design for PCR in real time amplification.

Every secondary response uses 2ng purified mRNA, carries out real time reaction according to standard pcr.For real-time detection, add SybrI dyestuff (MolecularProbes) with whole dilution factor 1/75000.Employing conventional instrument setting value increases (40 circulations) and detects in real time in Perkin-Elmer Biosystems PE7700 system.Use PE7700 Sequence Detector computed in software Ct value.Obtain several Ct values for each sample: for patient's sample, tumor Ct (CtT) value on candidate TAA and Normal Colon Ct (CtN) value of pairing, and for this group normal structure sample, the CtXY of each normal structure XY.Also calculate another Ct (CtA) of actin gene for all samples, as the internal reference thing of all samples.On the other hand, can adopt the amplification of Taqman probe monitoring PCR in real time.Employing conventional instrument setting value increases (40 circulations) and detects in real time in Perkin-Elmer Biosystems PE7700 system.Use PE7700 Sequence Detector computed in software Ct value.Obtain the Ct value of said target mrna (CtX) and actin mRNA (CtA) from each tissue sample.

Because the pcr amplification efficiency under common experiment condition is close to theoretical amplification efficiency, so 2 ^{(CtN/T/XY-CtA)}be the estimated value of the relative TAA transcriptional level of described sample, carry out standardized value with respect to actin transcriptional level.Therefore, numerical value is that the expression of 1 expression candidate antigens and actin is identical.

First the Normal Colon of the tumor colon to the biopsy from 12 patients and pairing carries out real-time PCR reactions.Then on 18 patients' altogether more fully data set, react (front 12 patients' data set is included).The data of 6 patients in these 18 patients of this data centralization are made with duplication.Also tested other 6 patients, result and front 18 patients' data merge.The results of statistical analysis that final merging value is carried out is shown in Table 3, and is illustrated in Fig. 1.

Also represent a series of 48 normal structure samples (normal structure of analyzing is shown in Table 3) of 29 kinds of different tissues according to same procedure test.Calculate as mentioned above TAA transcriptional level.Also according to this data set calculate overexpression candidate antigens patient ratio and with respect to the transcript overexpression meansigma methods of normal structure.The results are shown in Fig. 1.

Table 1:CASB7439 PCR in real time expression of results: 12 patients' data set

MRNA level is higher patient's percentage rate (positive patient) in the tumor colon of pairing	92％
		MRNA level is patient's percentage rate of at least high 3 times in the tumor colon of pairing	92％
MRNA level is patient's percentage rate of at least high 10 times in the tumor colon of pairing	92％
		MRNA level is patient's percentage rate of at least low 3 times in the tumor colon of pairing	8％
The Normal Colon mRNA horizontal average (using actin standardization) of pairing	0.0026
		The Normal Colon mRNA horizontal average (using actin standardization) of matching in positive patient	0.265
MRNA overexpression multiple meansigma methods	2028
		MRNA overexpression multiple intermediate value	115
Normal structure mRNA horizontal average	0.0079
		The horizontal intermediate value of normal structure mRNA	0.0016
Normal structure mRNA horizontal average	0.0064
		The horizontal intermediate value of normal structure mRNA	0.0017
Patient's percentage rate that mRNA level is higher than normal structure meansigma methods	92％
		MRNA level is than patient's percentage rate of high 10 times of normal structure meansigma methods	75％
Essential (non-dispensable) normal structure higher than the horizontal intermediate value of the mRNA of normal structure	Nothing

table 2:CASB7439 PCR in real time expression of results: 18 patients' data set

MRNA level is higher patient's percentage rate (positive patient) in the tumor colon of pairing	89％
		MRNA level is patient's percentage rate of at least high 3 times in the tumor colon of pairing	89％
MRNA level is patient's percentage rate of at least high 10 times in the tumor colon of pairing	78％
		MRNA level is patient's percentage rate of at least low 3 times in the tumor colon of pairing	5％
The Normal Colon mRNA horizontal average (using actin standardization) of pairing	0.005
		The Normal Colon mRNA horizontal average (using actin standardization) of matching in positive patient	0.152
MRNA overexpression multiple meansigma methods	1100
		MRNA overexpression multiple intermediate value	60
Normal structure mRNA horizontal average	0.0065
		The horizontal intermediate value of normal structure mRNA	0.0015
Normal structure mRNA horizontal average	0.005
		The horizontal intermediate value of normal structure mRNA	0.0015
Patient's percentage rate that mRNA level is higher than normal structure meansigma methods	94％
		MRNA level is than patient's percentage rate of high 10 times of normal structure meansigma methods	94％
The essential normal structure higher than the horizontal intermediate value of the mRNA of normal structure	Nothing

Table 3:CASB7439 PCR in real time expression of results: 24 patients' data set

CASB7439 transcript level high patient's percentage rate (positive patient) in tumor colon than in adjacent Normal Colon	92％
		CASB7439 transcript level positive patient percentage rate of at least high 10 times in tumor colon than in adjacent Normal Colon	75％
In the tumor colon transcription thing overexpression multiple meansigma methods of positive patient	1289
		CASB7439 transcript level patient percentage rate higher than normal structure meansigma methods in tumor colon	96％
MRNA level in tumor colon than patient's percentage rate of at least high 10 times of normal structure meansigma methods	62.5％
		Wherein CASB7439 transcript is expressed the normal structure that equals the tumor transcript level in tumor	Nothing

Also adopt Taqman method (as mentioned above), the tumor colon from 6 patient's biopsies and adjacent Normal Colon are carried out to real-time PCR reactions.Adopt three duplications to measure for each sample, further calculate by meansigma methods.The results are shown in Fig. 1.In addition also represent, 36 normal structure samples (referring to table 5) of 28 kinds of different tissues according to same procedure test.The results are shown in Fig. 2.

Table 4: the CASB7439 PCR in real time expression of results that adopts Taqman probe

From different patients' tumor sample number	6
		CASB7439 transcript level patient percentage rate (positive patient) higher than adjacent Normal Colon in tumor colon	100％
CASB7439 transcript level in tumor colon than the positive patient percentage rate of at least high 10 times of adjacent Normal Colon	83％
		In the tumor transcription thing overexpression multiple meansigma methods of positive patient	109
CASB7439 transcript level patient percentage rate higher than normal structure meansigma methods in tumor colon	100％
		MRNA level in tumor colon than patient's percentage rate of at least high 10 times of normal structure meansigma methods	100％
Wherein CASB7439 transcript is expressed the normal structure that equals the tumor transcript level in tumor	Nothing

Result clearly illustrates that, compares with all above-mentioned normal structures with adjacent Normal Colon, and CASB7439 transcript is overexpression in colorectum tumor.Compared with adjacent Normal Colon, the strong overexpression of CASB7439 transcript in more than 90% described patient's tumor.Average overexpression multiple in described tumor is at least 100.In addition, compared with other normal structure, CASB7439 transcript overexpression in more than 90% described patient's colon tumor, wherein the more than 60% overexpression multiple in the described patient of overexpression CASB7439 transcript is at least 10 times.

Table 5: for the normal structure catalog of CASB7439 transcript expression analysis

Tissue	Abbreviation
		Adrenal gland	Ad_G1
Aorta	Ao
		Bladder	B1
Bone marrow	Bo_Ma
		Brain	Bra
Cervix uteri	Ce
		Colon	Co
Fallopian tube	Fa_Tu
		Heart	He
Ileum	I1
		Kidney	Ki
Liver	Li
		Lung	Lu
Lymph node	Ly_No
		Esophagus	Oe
Parathyroid gland	Pa_Thy
		Rectum	Re
Skin	Sk
		Skeletal muscle	Sk_Mu
Small intestinal	Sm_In
		Spleen	Sp
Stomach	St
		Thyroid	Thy
Trachea	Tra
		Ovary	Ov
Placenta Hominis	P1
		Prostate	Pr
Testis	Te

embodiment 2

The differential screening of cDNA array

Complete the evaluation to the tumor-related gene in deduction cDNA library by differential screening.

From 100 μ l overnight culture, extract bacteria total DNA.With guanidinium isothiocyanate cracking antibacterial, use magnetic glass (Boehringer) to carry out affinity purification to DNA of bacteria.Reclaim plasmid Insert Fragment by Advantage pcr amplification (Clontech) from DNA of bacteria.Adopt Biomek 96 HDRT instruments (Beekman), by PCR product point sample to two nylon membrane, produce High Density cDNA array.Mottled cDNA irradiates and film covalent bond through UV.By first film with prepare from the tumor of single patient mix cDNA probe hybridization.What second film prepared with the Normal Colon from same patient of equivalent mixes cDNA probe hybridization.Prepare probe cDNA by pcr amplification as mentioned above, and adopt AlkPhos Direct System (Amersham) by its labelling.Hybridization conditions and strictly washing as described in AlkPhos Direct test kit.Hybridization probe detects with chemiluminescence.Measure the intensity for hybridization of every kind of cDNA fragment on two traces by film light densitometry or direct measuring method (BioRad Fluor-S Max).Calculate the ratio (T/N) of tumor intensity for hybridization with the normal intensity for hybridization of each gene, to evaluate the overexpression degree in tumor.Gene to remarkable overexpression in colon tumor carries out follow-up study.Significance is at random defined as a standard deviation of T/N frequency distribution.Adopt the RNA from multiple patient's donors (> 18), repeat differential screening experiment, to estimate the frequency of the tumor of overexpression in this patient colony.In addition, by DNA array and normal structure from except colon (referring to catalog above) mix cDNA probe hybridization, to measure the expression of described candidate gene in these tissues.

embodiment 3

DNA microarray

Detect the mrna expression profile of gene big collection thing in multiple samples with DNA microarray.Supplement the data that obtain by PCR in real time by this information, and the independent measurement of gene expression dose in tumor tissues and normal structure is provided.

The example of the current technology for generation of DNA microarray comprises 1) Affymetrix " GeneChip " array, wherein adopt photoetching process to synthesize the surperficial synthetic oligonucleotide at described chip by solid state chemistry, 2) DNA point sample (spotting) technology, wherein by robot, small size DNA solution is deposited to the surface of solid phase (for example glass), being then fixed.In both cases, by described chip and such as, from destination organization (normal structure, tumor etc..) extract and hybridize by radioactivity or with the cDNA of fluorescence reporter molecule labelling or cRNA.Make marker material and described chip hybridization, then use special scanners, measure the probe amount that each sequence is combined on described chip.Described experiment can be set up with single fluorescence reporter molecule (or radioactivity), or can carry out with two kinds of fluorescence reporter molecules.Under latter event, wherein a kind of reporter molecule labelling of the each use in described two samples.Then by these two sequence competitive hybridizations on sample and the described DNA chip of labelling.Determine the ratio of two kinds of fluorescence signals of each sequence on described chip.Calculate the relative abundance of transcript described in described two samples by this ratio.Detailed protocol can derive from many sources, comprises " DNA microarray: a kind of practical approach (DNA Microarrays:A practical approach).Schena M.Oxford UniversityPress 1999 " and WWW (http://cmgm.stanford.edu/pbrown/protocols/index.html), http://arrayit.com/DNA-Microarray-Protocols/) and many professional distributors (for example Affymetrix).

embodiment 5

RNA-DNA engram analysis

By the tumor colon cDNA of Advantage PCR (referring to above) the limited amount mixing of increasing and the Normal Colon cDNA of pairing.Also use the messenger RNA of same procedure amplification from multiple normal structures.By increased cDNA, (1 μ is electrophoresis on 1.2% agarose gel g), is then transferred on nylon membrane.By described film and the probe hybridization (AlkPhos Direct System) of preparing by candidate TAA cDNA fragment.RNA-DNA engram analysis provides about tumor tissues and normal structure transcription thing size, whether has the information of splicing variants and transcript abundance.

embodiment 6

Rna blot analysis

Use 1 μ g poly A+mRNA to produce RNA trace according to standard method.Adopt Ready-to-Go system (Pharmacia) to prepare radioactive probe.

embodiment 7

The experimental identification of full length cDNA sequence

Adopt Lambda ZapII system (Stratagene) to build colon tumor cDNA library from 5 μ g poly A+mRNA.The scheme providing is provided, just uses SuperscriptII (LifeTechnologies) to carry out reverse transcription step.Build oligomerization dT primer library and random primer library.For each screening in library, plating approximately 1.5 × 10 ⁶individual phagus liber.Plaque is transferred on nylon leaching film, and adopts the cDNA probe hybridization with AlkPhos Direct labelling.By chemiluminescence detection positive bacteriophage.Positive bacteriophage is cut from agar plate, eluting in 500 μ l SM buffer, by gene specific, PCR confirms.By removing in body, the bacteriophage conversion of eluting is become to strand M13 phage.Then by colibacillary infection, described bacteriophage conversion is become to double-stranded plasmid DNA.By infected antibacterial plating, then carry out second with described cDNA probe and take turns screening.Go out plasmid DNA from positive bacteria clone purification, then to two chain order-checkings.

In the time that full-length gene can not directly obtain from cDNA library, adopt RACE technology (Marathon Kit, ClonTech) to separate and lose sequence.The method depends on mRNA reverse transcription is become to double-stranded cDNA, and joint is connected with two ends of cDNA, adopts the required end of the primer amplified cDNA of gene-specific primer and joint oligonucleotide.MarathonPCR product cloning, in plasmid (pCRII-TOPO, InVitrogen), is then checked order.

Use the method to obtain the polynucleotide of SEQ ID NO:1.

embodiment 8.

EST profile

A kind of complementarity method that experiment antigen tissue expression characterizes is seeker's est database.EST (" expressed sequence tag ") is the small fragment of the cDNA for preparing of the mRNA aggregation that extracts from particular organization or cell line.Such data base provides a large amount of people EST (2 10 that organizes library (comprising the tumor tissues from the disease of all kinds and stadium) from thousands of kinds of cDNA at present ⁶).By means of information tool (Blast), carry out the comparison search of CASB7439 sequence, to more in depth understand tissue expression.

The EST of CASB7439 distributes

EST GenBank registration number	EST cDNA organizes library
		C00634	Adult (K.Okubo)
AA468668	NCI_CGAP_Co3
		AA565752	NCI_CGAP_Co11
AA565766	NCI_CGAP_Co11
		AA565767	NCI_CGAP_Co11
AI337239	NCI_CGAP_Co16
		AI337448	NCI_CGAP_Co16
AI393930	NCI_CGAP_CLL1
		AI473673	NCI_CGAP_Co14
AI632444	NCI_CGAP_GC6
		AI861937	NCI_CGAP_Co16
AI825214	NCI_CGAP_GC6
		AW080652	NCI_CGAP_Co19
AW083899	NCI_CGAP_Co19
		AW206058	NCI_CGAP_Sub3
AW237006	NCI_CGAP_GC6
		AW364626	DT0036
AW449612	NCI_CGAP_Sub5

These EST and CASB7439 Perfect Matchings.This table comprises 9 EST from 4 different tumor colons libraries, 1 EST from 1 Normal Colon library, 3 EST from 1 tumor sexual cell library, 1 EST from 1 chronic lymphocytic leukemia cell library, 2 EST from 2 mixed rumour libraries, 2 EST from UNKNOWN TYPE library.This clearly illustrates that, with expection, compared with normal structure, CASB7439 is overexpression in tumor tissues, especially overexpression in colorectum tumor tissues.

embodiment 9:

The expression of 9.1 tumor specific antigens and purification

Adopt and in microbial hosts, express or transcribe in vitro/translate, produce for vaccine object antigen of the present invention and produce protein fragments or intact proteins, for fast purifying and produce by immunohistochemical method and identify that natural expressing protein or purification follow the tracks of required antibody.

Can for example, in two kinds of microbial hosts-escherichia coli and yeast (saccharomyces cerevisiae (Saccharomyces cerevisiar) or pichia pastoris phaff (Pichia pastoris)), express recombiant protein.This allows to select to have the expression system of best features, for producing this specific antigen.Generally speaking, allow recombinant antigen at expression in escherichia coli, and allow reagent albumen express in yeast.

First expression strategy comprises the primary structure that designs recombinant antigen.Generally will express fusion partner (EFP) and be placed in N-terminal, to improve expression, described expression fusion partner also can comprise region-Immune Fusion gametophyte (IFP) that can be used for regulating described antigen immune originality characteristic.In addition, comprise and can be used for contributing to the affinity fusion partner (AFP) that is further purified at C-end.

As mentioned above, can compare evaluation to several constructs:

For quick expression and purification and produce the antibody for CASB7439, suggestion in escherichia coli, produce have NS1 as EFP and histidine tail the total length CASB7439 albumen as AFP.

Therefore, two kinds of constructs are proposed:

Construct 1: with the total length wild type CASB7439 cDNA (SEQ ID NO:8) of NS1 cDNA (as EFP) and histidine tail code cDNA (as AFP) fusion.Coded fusion rotein sequence is SEQ ID NO:10.

Construct 2: with the total length saltant type CASB7439 cDNA (SEQ ID NO:9) of NS1 cDNA (as EFP) and histidine tail code cDNA (as AFP) fusion.Suggestion has by e. coli codon in this construct selects specific codon to replace front 50 codons of natural CASB7439 cDNA, to strengthen the expression potentiality of CASB7439 in its escherichia coli host.Coded fusion rotein sequence is SEQ ID NO:10.

Described CASB7439 protein design is as follows:

" NS1 " is the N-end fragment (80 aminoacid) of influenza virus protein NS1." HIS " is poly histidine tail.

Recombinant bacterial strain used is AR58: be derived from the disguised λ lysogen of N99 and cI857, N99 is gal E::Tn 10, Δ-8 (chlD-pgl), Δ-H1 (cro-chlA), N ⁺(Proc.Natl.Acad.Sci.USA the 82nd volume, 88-92 page, in January, 1985 Biochemistry).

In the time that recombinant bacterial strain can obtain, further predict that by evaluation expression level and by analyzing the behavior of crude extract the dissolubility of described albumen characterizes recombinant products.

On suitable culture medium, grow and induce after expression of recombinant proteins, by SDS-PAGE analyzing total extract.Recombiant protein is manifested on dyeing gel, and right and use specific antibody is identified by western blot analysis.

Plasmid:

Title: TCM 281 pRIT..15143

Replicon: pMB1

Select: Kan

Promoter: PL long

Insert Fragment: NS1-C74-39-His

Express recombiant protein with construct 1:

Allow antibacterial in the μ g/ml Kan of LB culture medium+50 in 30 ℃ of growths

In the time that culture reaches OD=0.5 (620nm), culture is heated to 39 ℃ at the most,

Induce after 5 hours harvesting

The preparation of extract:

Cell concentration: .50X. is complete at buffer PBS+ ... in

Broken: French press 3X

Centrifugal: in 14000t 30 minutes

Remarks: > 90% in the supernatant of cell extract

By cell extract electrophoresis on 12.5%SDS PAGE, use subsequently Coomassie blue stain.Also use the commercially available monoclonal antibody for poly histidine tail (Quiagen) to carry out western blot analysis.Gained gel (Fig. 3 and Fig. 4) shows that this albumen is expressed, and manifests in cell extract supernatant.

Purification schemes adopts the classical way existing based on histidine affinity tail in recombiant protein.In a model experiment, filter broken antibacterial, then cell-free extract is loaded onto to the ionic metal affinity column (IMAC that retains specifically described recombiant protein; Ni ⁺⁺nTA, derives from Qiagen) on.In phosphate buffer, with the albumen retaining described in 0-500mM imidazoles gradient (may detergent exist under) eluting.

To derive from the supernatant of gathering in the crops culture at 6M carbamide, 100mM NaH ₂pO ₄, degeneration in 10mMTris, pH8, be then loaded under the following conditions chromatographic column IMAC Qiagen NTANi ⁺⁺:

Level pad: NaH ₂pO ₄100mM pH8

Tris 10mM

Carbamide 6M

Sample: carbamide 6M, 100mM NaH ₂pO ₄, supernatant in 10mM Tris

Lavation buffer solution: 1) NaH ₂pO ₄100mM pH8

Tris 10mM

Carbamide 6M

Imidazoles 25mM

2)NaH ₂PO ₄ 100mM pH8

Tris 10mM

Carbamide 6mM

Imidazoles 50mM

Elution buffer: NaH ₂pO ₄100mM pH5.5

Tris 10mM

Carbamide 6M

Imidazoles 500mM

Eluted protein in 500mM imidazoles+6M carbamide is dialysed under the following conditions:

-PBS PH7.2+sarkosyl 0.5%+4M carbamide

-the same, in 2M carbamide 2 hours

-the same, in 0M carbamide 2 hours

By freezing final material preservation.Protein content adopts Lowry protein determination to come quantitatively (0.9mg/1.2ml).By evaluating purity (Fig. 5) with the 12.5%PAGE SDS of Coomassie blue stain, by Western blotting, adopt anti-poly histidine monoclonal antibody to check exist (Fig. 6) of recombiant protein.

To the comparative evaluation of multi-form expressed antigen, will make it possible to select the most promising material standed for, then use it for and be further purified and immunological evaluation.

The generation of 9.2 antibody and immunohistochemistry

Can produce immunology instrument with the albumen of a small amount of purification relatively, so that

A) in normal or cancerous tissue section, detect described expression by immunohistochemistry;

B) detect described expression, and during purge process, follow the tracks of described albumen (ELISA/ Western blotting); Or

C) evaluation/quantitative described purifying protein (ELISA).

9.2.1 polyclonal antibody:

immunity

Rabbit is formulated in albumen in adjuvant 3D-MPL/QS21 with 3 weekly interval intramusculars (I.M.) immunity 3 times with 100 μ g.Latter 3 weeks of each immunity, takes a blood sample, and then uses described albumen as envelope antigen, according to standard method, by ELISA, estimates the antibody titer in described serum.

ELISA

96 hole microtest plates (maxisorb Nunc) are coated with and are spent the night in 4 ℃ with 5 μ g albumen.After saturated 1 hour, add the rabbit anteserum (originate in 1/10) of serial dilution to reach 1 hour 30 minute in 37 ℃ in 37 ℃ with 1%PBS NCS 1%.With after PBS tween washing 3 times, add anti-rabbit biotinylation antiserum (Amersham) (1/5000).Washing culture plate, then adds peroxidase coupling Succ-PEG-DSPE (1/5000) to reach 30 minutes in 37 ℃.After washing, add 50 μ l TMB (BioRad) to reach 7 minutes, then use 0.2M H ₂sO ₄cessation reaction.Can measure OD at 450nm, and calculate mid point dilution factor with SoftmaxPro.

9.2.2 monoclonal antibody

immunity

With 5 μ g purifying proteins to 5 BALB/c mouse with 3 weekly intervals immunity 3 times.After latter 14 days of the 2nd immunity and the 3rd immunity, within 1 week, carry out blood-letting.Make envelope antigen with purifying protein, test described serum by Elisa.According to these results (mid point dilution factor > 10000), select 1 mice for merging.

fusion/HAT selects

According to standard method, use 40%PEG and 5%DMSO, splenocyte and SP2/0 myeloma cell are merged.Then with 2.5 × 10 ⁴-10 ⁵cell is inoculated in 96 orifice plates by cells/well, then in HAT culture medium, selects resistance clone.Test the content of specific antibody in these hybridoma supernatant, in the time that hybridoma supernatant is positive, the limiting dilution that is carried out 2 circulations.Take turns after screening through 2, select 3 hybridomas to produce for ascites.

9.2.3 immunohistochemistry

In the time that antibody can obtain, immunostaining is carried out in normal tissue section or cancerous tissue section, to determine:

◇ for normal structure in cancerous tissue the expression of antigen of the present invention or

◇ expresses the ratio of a certain type cancer of described antigen

Whether other cancer type is also expressed described antigen to ◇

◇ expresses the cell proportion of described antigen in a kind of cancerous tissue

tissue sample preparation

After dissection, tissue sample is fixed on cork dish with OCT compound, then in previous quick freezing in supercool isopentane in liquid nitrogen (160 ℃).Use before always by described frozen block-70 ℃ of preservations.7-10 μ m section completes in cryostat chamber (20 ,-30 ℃).

dyeing

Tissue slice is dried to 5 minutes under room temperature (RT), fixes 10 minutes under room temperature in acetone, after drying, then uses PBS 0.5%BSA 5% serum saturated.In room temperature after lower 30 minutes, directly or indirectly dye with antigen-specific antibodies.Direct staining produces the better but lower dyeing of intensity of specificity, and dyeing produces the higher but dyeing that specificity is lower of intensity indirectly.

The analysis of the 9.3 people's cellullar immunologic responses for antigen of the present invention

By making in vitro human T-cell contact antigen, can evaluate the immune-related of antigen of the present invention.All T cell lymphocyte systems and dendritic cell all derive from the PBMC (peripheral blood lymphocytes) of healthy donors (being preferably HLA-A2 hypotype).HLA-A2.1/Kb transgene mouse model is also used for screening HLA-A2.1 peptide.

By stimulated in vitro weekly, produce and maintain newfound antigenic specificity CD8 ⁺t cell line.Adopt standard method, test described CD8 ⁺described antigen or the lytic activity of antigen derived peptide and the generation of γ-IFN are replied by system.

Produce described CD8 with two kinds of strategies ⁺t cell line: a kind of a kind of method and method based on complete genome based on peptide.Two kinds of methods all require by the full-length cDNA of the correct frame of new discovery antigen or in proper delivery system clone or be used for predicting the sequence of HLA binding peptide.

based on the method for peptide

General introduction ground is said, with the HLA-A2 peptide immune transgenic mice containing adjuvant, can not induce CD8 ⁺reply those peptides of (determining according to the effective cracking of self splenocyte of peptide burst process) further analyzes in robot system.

People's dendritic cell (cultivating according to described methods such as Romani) carry out burst process with peptide, and are used for stimulating CD8 ⁺the T cell (passing through Facs) of classification.After stimulating weekly for several times, first at the described CD8 of the upper test of self BLCL (EBV-B transformation cell lines) of peptide burst process ⁺system.In order to confirm the described peptide of correct processing in vivo, at the described CD8 of the upper test of tumor cell (LnCap, the Skov3 of HLA-A2 transfection or CAMA tumor cell) of cDNA transfection ⁺system.

based on the method for complete genome

Make CD8 ⁺t cell line contact antigen, then the dendritic cell of fibroblast, recombinant poxvirus or the adenovirus infection of the B7.1 transfection of the dendritic cell of use particle gun transfection, retrovirus retrovirus transduction stimulate.The cell of viral infection is antigen-presenting peptide very effectively, because described antigen is with high level expression, and can only use once, to avoid the undue growth of viral T cell line.

Change after stimulation, as mentioned above, on the tumor cell of cDNA transfection, test described CD8 ⁺system.Measure peptide specific and homogeneity to confirm immunological test.

CD4 ⁺t cell response

Equally, also can evaluate CD4 ⁺t cellullar immunologic response.Adopt and load in order to stimulate the restructuring purifying protein of T cell or the dendritic cell of peptide to produce specific C D4 ⁺t cell.

prediction is in conjunction with the allelic epi-position of HLA (nine aggressiveness and ten aggressiveness):

According to Parker algorithm (Parker, K.C., the independent combination of M.A.Bednarek and the indivedual peptide strands of J.E.Coligan.1994. basis is by the potential graduate scheme J.Immunol.152:163 of HLA-A2 binding peptide and http://bimas.dcrt.nih.gov/molbio/hla_bind) or Rammenscc method (Rammensee, Friede, Stevanovic, MHC part and peptide motif: the first list, Immunogenetics 41,178-228,1995; Rammensee, Bachmann, Stevanovic:MHC part and peptide motif.Landes Bioscience 1997 and http: // 134.2.96.221/scripts/hlaserver.dll/home.htm), prediction HLAI class peptide binding sequence.Then screening peptide in HLA-A2.1/Kb transgene mouse model (Vitiello etc.).

Use Tepitope algorithm, score and be set to 6 (Sturniolo, Hammer etc., Nature Biotechnology.1999.17 with cut-off; 555-561), prediction HLAII class peptide binding sequence.

Following table has been collected the epitope sequences of I class and the prediction of II class:

°: the estimation of the molecular dissociation half-life of containing described subsequence.

°: the estimation of the molecular dissociation half-life of containing described subsequence.

°: the estimation of the molecular dissociation half-life of containing described subsequence.

°: the estimation of the molecular dissociation half-life of containing described subsequence.

°: the estimation of the molecular dissociation half-life of containing described subsequence.

°: the estimation of the molecular dissociation half-life of containing described subsequence.

Sequence information

Sequence table

<110> Smithkline Beecham Biologicals

(SmithKline Beecham Biologicals s.a.)

<120> noval chemical compound

<130>BC45300

<160>33

<170>FastSEQ for Windows Version 3.0

<210>1

<211>1791

<212>DNA

The <213> mankind

<400>1

gtaccttgct ttgggggcgc actaagtacc tgccgggagc agggggcgca ccgggaactc 60

gcagatttcg ccagttgggc gcactgggga tctgtggact gcgtccgggg gatgggctag 120

ggggacatgc gcacgctttg ggccttacag aatgtgatcg cgcgaggggg agggcgaagc 180

gtggcgggag ggcgaggcga aggaaggagg gcgtgagaaa ggcgacggcg gcggcgcgga 240

ggagggttat ctatacattt aaaaaccagc cgcctgcgcc gcgcctgcgg agacctggga 300

gagtccggcc gcacgcgcgg gacacgagcg tcccacgctc cctggcgcgt acggcctgcc 360

accactaggc ctcctatccc cgggctccag acgacctagg acgcgtgccc tggggagttg 420

cctggcggcg ccgtgccaga agcccccttg gggcgccaca gttttccccg tcgcctccgg 480

ttcctctgcc tgcaccttcc tgcggcgcgc cgggacctgg agcgggcggg tggatgcagg 540

cgcgatggac ggcggcacac tgcccaggtc cgcgccccct gcgccccccg tccctgtcgg 600

ctgcgctgcc cggcggagac ccgcgtcccc ggaactgttg cgctgcagcc ggcggcggcg 660

accggccacc gcagagaccg gaggcggcgc agcggccgta gcgcggcgca atgagcgcga 720

gcgcaaccgc gtgaagctgg tgaacttggg cttccaggcg ctgcggcagc acgtgccgca 780

cggcggcgcc agcaagaagc tgagcaaggt ggagacgctg cgctcagccg tggagtacgt 840

ccgcgcgctg cagcgcctgc tggccgagca cgacgccgtg cgcaacgcgc tggcgggagg 900

gctgaggccg caggccgtgc ggccgtctgc gccccgcggg ccgccaggga ccaccccggt 960

cgccgcctcg ccctcccgcg cttcttcgtc cccgggccgc gggggcagct cggagcccgg 1020

ctccccgcgt tccgcctact cgtcggacga cagcggctgc gaaggcgcgc tgagtcctgc 1080

ggagcgcgag ctactcgact tctccagctg gttagggggc tactgagcgc cctcgaccta 1140

tgagcctcag ccccggaagc cgagcgagcg gccggcgcgc tcatcgccgg ggagcccgcc 1200

a ggtggaccg gcccgcgctc cgcccccagc gagccgggga cccacccacc accccccgca 1260

ccgccgacgc cgcctcgttc gtccggccca gcctgaccaa tgccgcggtg gaaacgggct 1320

tggagctggc cccataaggg ctggcggctt cctccgacgc cgcccctccc cacagcttct 1380

cgactgcagt ggggcggggg gcaccaacac ttggagattt ttccggaggg gagaggattt 1440

tctaagggca cagagaatcc attttctaca cattaacttg agctgctgga gggacactgc 1500

tggcaaacgg agacctattt ttgtacaaag aacccttgac ctggggcgta ataaagatga 1560

cctggacccc tgcccccact atctggagtt ttccatgctg gccaagatct ggacacgagc 1620

agtccctgag gggcggggtc cctggcgtga ggcccccgtg acagcccacc ctggggtggg 1680

tttgtgggca ctgctgctct gctagggaga agcctgtgtg gggcacacct cttcaaggga 1740

gcgtgaactt tataaataaa tcagttctgt ttaaaaaaaa aaaaaaaaaa a 1791

<210>2

<211>193

<212>PRT

The <213> mankind

<400>2

Met Asp Gly Gly Thr Leu Pro Arg Ser Ala Pro Pro Ala Pro Pro Val

1 5 10 15

Pro Val Gly Cys Ala Ala Arg Arg Arg Pro Ala Ser Pro Glu Leu Leu

20 25 30

Arg Cys Ser Arg Arg Arg Arg Pro Ala Thr Ala Glu Thr Gly Gly Gly

35 40 45

Ala Ala Ala Val Ala Arg Arg Asn Glu Arg Glu Arg Asn Arg Val Lys

50 55 60

Leu Val Asn Leu Gly Phe Gln Ala Leu Arg Gln His Val Pro His Gly

65 70 75 80

Gly Ala Ser Lys Lys Leu Ser Lys Val Glu Thr Leu Arg Ser Ala Val

85 90 95

Glu Tyr Ile Arg Ala Leu Gln Arg Leu Leu Ala Glu His Asp Ala Val

100 105 110

Arg Asn Ala Leu Ala Gly Gly Leu Arg Pro Gln Ala Val Arg Pro Ser

115 120 125

Ala Pro Arg Gly Pro Pro Gly Thr Thr Pro Val Ala Ala Ser Pro Ser

130 135 140

Arg Ala Ser Ser Ser Pro Gly Arg Gly Gly Ser Ser Glu Pro Gly Ser

145 150 155 160

Pro Arg Ser Ala Tyr Ser Ser Asp Asp Ser Gly Cys Glu Gly Ala Leu

165 170 175

Ser Pro Ala Glu Arg Glu Leu Leu Asp Phe Ser Ser Trp Leu Gly Gly

180 185 190

Tyr

<210>3

<211>262

<212>PRT

The <213> mankind

<400>3

Met Ser Ala Pro Ala Ala Arg Ser Ala Ser Gly Ala Glu Ala His Arg

1 5 10 15

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

20 25 30

Arg Ala Pro Gln Asp Ser Ala Arg Leu Arg Ser Arg Cys Arg Pro Thr

35 40 45

Ser Arg Arg Asn Ala Gly Ser Arg Ala Pro Ser Cys Pro Arg Gly Pro

50 55 60

Gly Thr Lys Lys Arg Gly Arg Ala Arg Arg Arg Pro Gly Trp Ser Leu

65 70 75 80

Ala Ala Arg Gly Ala Gln Thr Ala Ala Arg Pro Ala Ala Ser Ala Leu

85 90 95

Pro Pro Ala Arg Cys Ala Arg Arg Arg Ala Arg Pro Ala Gly Ala Ala

100 105 110

Ala Arg Gly Cys Thr Pro Arg Leu Ser Ala Ala Ser Pro Pro Cys Ser

115 120 125

Ala Ser Cys Trp Arg Arg Arg Ala Ala Arg Ala Ala Ala Ala Pro Gly

130 135 140

Ser Pro Ser Ser Pro Ala Ser Arg Gly Cys Ala Arg Ala His Cys Ala

145 150 155 160

Ala Leu Arg Pro Leu Arg Arg Leu Arg Ser Leu Arg Trp Pro Val Ala

165 170 175

Ala Ala Gly Cys Ser Ala Thr Val Pro Gly Thr Arg Val Ser Ala Gly

180 185 190

Gln Arg Ser Arg Gln Gly Arg Gly Ala Gln Gly Ala Arg Thr Trp Ala

195 200 205

Val Cys Arg Arg Pro Ser Arg Leu His Pro Pro Ala Arg Ser Arg Ser

210 215 220

Arg Arg Ala Ala Gly Arg Cys Arg Gln Arg Asn Arg Arg Arg Arg Gly

225 230 235 240

Lys Leu Trp Arg Pro Lys Gly Ala Ser Gly Thr Ala Pro Pro Gly Asn

245 250 255

Ser Pro Gly His Ala Ser

260

<210>4

<211>1830

<212>DNA

The <213> mankind

<400>4

gtaccttgct ttgggggcgc actaagtacc tgccgggagc agggggcgca ccgggaactc 60

gcagatttcg ccagttgggc gcactgggga tctgtggact gcgtccgggg gatgggctag 120

ggggacatgc gcacgctttg ggccttacag aatgtgatcg cgccgagggg gagggccgaa 180

gcgtggcggg agggcgaggc gaaggaagga gggcgtgaga aaggcgacgg cggcggcgcg 240

gaggagggtt atctatacat ttaaaaacca gccgcctgcg ccgcgcctgc ggagacctgg 300

gagagtccgg ccgcacgcgc gggacacgag cgtcccacgc tccctggcgc gtacggcctg 360

ccaccactag gcctcctatc cccgggctcc agacgaccta ggacgcgtgc cctggggagt 420

tgcctggcgg cgccgtgcca gaagccccct tggggcgcca cagttttccc cgtcgcctcc 480

ggttcctctg cctgcacctt cctgcggcgc gccgggacct ggagcgggcg ggtggatgca 540

ggcgcgatgg acggcggcac actgcccagg tccgcgcccc ctgcgccccc cgtccctgtc 600

ggctgcgctg cccggcggag acccgcgtcc ccggaactgt tgcgctgcag ccggcggcgg 660

cgaccggcca ccgcagagac cggaggcggc gcagcggccg tagcgcggcg caatgagcgc 720

gagcgcaacc gcgtgaagct ggtgaacttg ggcttccagg cgctgcggca gcacgtgccg 780

cacggcggcg ccagcaagaa gctgagcaag gtggagacgc tgcgctcagc cgtggagtac 840

atccgcgcgc tgcagcgcct gctggccgag cacgacgccg tgcgcaacgc gctggcggga 900

gggctgaggc cgcaggccgt gcggccgtct gcgccccgcg ggccgccagg gaccaccccg 960

gtcgccgcct cgccctcccg cgcttcttcg tccccgggcc gcgggggcag ctcggagccc 1020

ggctccccgc gttccgccta ctcgtcggac gacagcggct gcgaaggcgc gctgagtcct 1080

gcggagcgcg agctactcga cttctccagc tggttagggg gctactgagc gccctcgacc 1140

taataagcct caagccccgg aaacccgagc gaacgggccg gcgcgcttca tcgccgggga 1200

agcccgccaa ggtggaccgg gcccgcgctc cgcccccagc gagccgggga cccacccacc 1260

accccccgca ccgccgacgc cgcctcgttc gtccggccca gcctgaccaa tgccgcggtg 1320

gaaacgggct tggagctggc cccataaggg ctggcggctt cctccgacgc cgcccctccc 1380

cacagcttct cgactgcagt ggggcggggg gcaccaacac ttggagattt ttccggaggg 1440

gagaggattt tctaagggca cagagaatcc attttctaca cattaacttg agctgctgga 1500

gggacactgc tggcaaacgg agacctattt ttgtacaaag aacccttgac ctggggcgta 1560

ataaagatga cctggacccc tgcccccact atctggagtt ttccatgctg gccaagatct 1620

ggacacgagc agtccctgag gggcggggtc cctggcgtga ggcccccgtg acagcccacc 1680

ctggggtggg tttgtgggca ctgctgctct gctagggaga agcctgtgtg gggcacacct 1740

cttcaaggga gcgtgaactt tataaataaa tcagttctgt ttaaaaaaaa aaaaaaaaaa 1800

aaaaccgagg gggggcccgg agccaacaaa 1830

<210>5

<211>587

<212>DNA

The <213> mankind

<400>5

ggtaaacaga actgatttat ttataaagtt cacgctccct tgaagaggtg tgccccacac 60

aggcttctcc ctagcagagc agcagtgccc acaaacccac cccagggtgg gctgtcacgg 120

gggcctcacg ccagggaccc cgcccctcag ggactgctcg tgtccagatc ttggccagca 180

tggaaaactc cagatagtgg gggcaggggt ccaggtcatc tttattacgc cccaggtcaa 240

gggttctttg tacaaaaata ggtctccgtt tgccagcagt gtccctccag cagctcaagt 300

taatgtgtag aaaatggatt ctctgtgccc ttagaaaatc ctctcccctc cggaaaaatc 360

tccaagtgtt ggtgcccccc gccccactgc agtcgagaag ctgtggggag gggcggcgtc 420

ggaggaagcc gcagcccatt atggggccag ctccaagccc gtttccaccg cggcattggt 480

caggctgggc ggacgaacga ggcggcgtcg gcggtgcggg gggtggtggg tgggtccccg 540

gctcgctggg ggcggagcag cgggccggtc cacctggcgg gctcccc 587

<210>6

<211>1791

<212>DNA

The <213> mankind

<400>6

tttttttttt ttttttttta aacagaactg atttatttat aaagttcacg ctcccttgaa 60

gaggtgtgcc ccacacaggc ttctccctag cagagcagca gtgcccacaa acccacccca 120

gggtgggctg tcacgggggc ctcacgccag ggaccccgcc cctcagggac tgctcgtgtc 180

cagatcttgg ccagcatgga aaactccaga tagtgggggc aggggtccag gtcatcttta 240

ttacgcccca ggtcaagggt tctttgtaca aaaataggtc tccgtttgcc agcagtgtcc 300

ctccagcagc tcaagttaat gtgtagaaaa tggattctct gtgcccttag aaaatcctct 360

cccctccgga aaaatctcca agtgttggtg ccccccgccc cactgcagtc gagaagctgt 420

ggggaggggc ggcgtcggag gaagccgcca gcccttatgg ggccagctcc aagcccgttt 480

ccaccgcggc attggtcagg ctgggccgga cgaacgaggc ggcgtcggcg gtgcgggggg 540

tggtgggtgg gtccccggct cgctgggggc ggagcgcggg ccggtccacc tggcgggctc 600

cccggcgatg agcgcgccgg ccgctcgctc ggcttccggg gctgaggctc ataggtcgag 660

ggcgctcagt agccccctaa ccagctggag aagtcgagta gctcgcgctc cgcaggactc 720

agcgcgcctt cgcagccgct gtcgtccgac gagtaggcggaacgcgggga gccgggctcc 780

gagctgcccc cgcggcccgg ggacgaagaa gcgcgggagg gcgaggcggc gaccggggtg 840

gtccctggcg gcccgcgggg cgcagacggc cgcacggcct gcggcctcag ccctcccgcc 900

agcgcgttgc gcacggcgtc gtgctcggcc agcaggcgct gcagcgcgcg gatgtactcc 960

acggctgagc gcagcgtctc caccttgctc agcttcttgc tggcgccgcc gtgcggcacg 1020

tgctgccgca gcgcctggaa gcccaagttc accagcttca cgcggttgcg ctcgcgctca 1080

ttgcgccgcg ctacggccgc tgcgccgcct ccggtctctg cggtggccgg tcgccgccgc 1140

cggctgcagc gcaacagttc cggggacgcg ggtctccgcc gggcagcgca gccgacaggg 1200

acggggggcg cagggggcgc ggacctgggc agtgtgccgc cgtccatcgc gcctgcatcc 1260

acccgcccgc tccaggtccc ggcgcgccgc aggaaggtgc aggcagagga accggaggcg 1320

acggggaaaa ctgtggcgcc ccaagggggc ttctggcacg gcgccgccag gcaactcccc 1380

agggcacgcg tcctaggtcg tctggagccc ggggatagga ggcctagtgg tggcaggccg 1440

tacgcgccag ggagcgtggg acgctcgtgt cccgcgcgtg cggccggact ctcccaggtc 1500

tccgcaggcg cggcgcaggc ggctggtttt taaatgtata gataaccctc ctccgcgccg 1560

ccgccgtcgc ctttctcacg ccctccttcc ttcgcctcgc cctcccgcca cgcttcgccc 1620

tccccctcgc gcgatcacat tctgtaaggc ccaaagcgtg cgcatgtccc cctagcccat 1680

cccccggacg cagtccacag atccccagtg cgcccaactg gcgaaatctg cgagttcccg 1740

gtgcgccccc tgctcccggc aggtacttag tgcgccccca aagcaaggta c 1791

<210>7

<211>361

<212>PRT

The <213> mankind

<400>7

Met Cys Arg Lys Trp Ile Leu Cys Ala Leu Arg Lys Ser Ser Pro Leu

1 5 10 15

Arg Lys Asn Leu Gln Val Leu Val Pro Pro Ala Pro Leu Gln Ser Arg

20 25 30

Ser Cys Gly Glu Gly Arg Arg Arg Arg Lys Pro Pro Ala Leu Met Gly

35 40 45

Pro Ala Pro Ser Pro Phe Pro Pro Arg His Trp Ser Gly Trp Ala Gly

50 55 60

Arg Thr Arg Arg Arg Arg Arg Cys Gly Gly Trp Trp Val Gly Pro Arg

65 70 75 80

Leu Ala Gly Gly Gly Ala Arg Ala Arg Ser Thr Leu Ala Gly Phe Pro

85 90 95

Gly Asp Glu Ala Arg Arg Pro Val Arg Ser Gly Phe Arg Gly Leu Arg

100 105 110

Leu Ile Arg Ser Arg Ala Leu Ser Ser Pro Leu Thr Ser Trp Arg Ser

115 120 125

Ara Val Ala Arg Ala Pro Gln Asp Ser Ala Arg Leu Arg Ser Arg Cys

130 135 140

Arg Pro Thr Ser Arg Arg Asn Ala Gly Ser Arg Ala Pro Ser Cys Pro

145 150 155 160

Arg Gly Pro Gly Thr Lys Lys Arg Gly Arg Ala Arg Arg Arg Pro Gly

165 170 175

Trp Ser Leu Ala Ala Arg Gly Ala Gln Thr Ala Ala Arg Pro Ala Ala

180 185 190

Ser Ala Leu Pro Pro Ala Arg Cys Ala Arg Arg Arg Ala Arg Pro Ala

195 200 205

Gly Ala Ala Ala Arg Gly Cys Thr Pro Arg Leu Ser Ala Ala Ser Pro

210 215 220

Pro Cys Ser Ala Ser Cys TrpArg Arg Arg Ala Ala Arg Ala Ala Ala

225 230 235 240

Ala Pro Gly Ser Pro Ser Ser Pro Ala Ser Arg Gly Cys Ala Arg Ala

245 250 255

His Cys Ala Ala Leu Arg Pro Leu Arg Arg Leu Arg Ser Leu Arg Trp

260 265 270

Pro Val Ala Ala Ala Gly Cys Ser Ala Thr Val Pro Gly Thr Arg Val

275 280 285

Ser Ala Gly Gln Arg Ser Arg Gln Gly Arg Gly Ala Gln Gly Ala Arg

290 295 300

Thr Trp Ala Val Cys Arg Arg Pro Ser Arg Leu His Pro Pro Ala Arg

305 310 315 320

Ser Arg Ser Arg Arg Ala Ala Gly Arg Cys Arg Gln Arg Asn Arg Arg

325 330 335

Arg Arg Gly Lys Leu Trp Arg Pro Lys Gly Ala Ser Gly Thr Ala Pro

340 345 350

Pro Gly Asn Ser Pro Gly His Ala Ser

355 360

<210>8

<211>849

<212>DNA

<213> influenza virus and the mankind

<400>8

atggatccaa acactgtgtc aagctttcag gtagattgct ttctttggca tgtccgcaaa 60

cgagttgcag accaagaact aggtgatgcc ccattccttg atcggcttcg ccgagatcag 120

aaatccctaa gaggaagggg cagcaccctc ggtctggaca tcgagacagc cacacgtgct 180

ggaaagcaga tagtggagcg gattctgaaa gaagaatccg atgaggcact taaaatgacc 240

atggacggcg gcacactgcc caggtccgcg ccccctgcgc cccccgtccc tgtcggctgc 300

gctgcccggc ggagacccgc gtccccggaa ctgttgcgct gcagccggcg gcggcgaccg 360

gccaccgcag agaccggagg cggcgcagcg gccgtagcgc ggcgcaatga gcgcgagcgc 420

aaccgcgtga agctggtgaa cttgggcttc caggcgctgc ggcagcacgt gccgcacggc 480

ggcgccagca agaagctgag caaggtggag acgctgcgct cagccgtgga gtacatccgc 540

gcgctgcagc gcctgctggc cgagcacgac gccgtgcgca acgcgctggc gggagggctg 600

aggccgcagg ccgtgcggcc gtctgcgccc cgcgggccgc cagggaccac cccggtcgcc 660

gcctcgccct cccgcgcttc ttcgtccccg ggccgcgggg gcagctcgga gcccggctcc 720

ccgcgttccg cctactcgtc ggacgacagc ggctgcgaag gcgcgctgag tcctgcggag 780

cgcgagctac tcgacttctc cagctggtta gggggctaca ctagtggcca ccatcaccat 840

caccattaa 849

<210>9

<211>849

<212>DNA

<213> influenza virus and the mankind

<400>9

atggatccaa acactgtgtc aagctttcag gtagattgct ttctttggca tgtccgcaaa 60

cgagttgcag accaagaact aggtgatgcc ccattccttg atcggcttcg ccgagatcag 120

aaatccctaa gaggaagggg cagcaccctc ggtctggaca tcgagacagc cacacgtgct 180

ggaaagcaga tagtggagcg gattctgaaa gaagaatccg atgaggcact taaaatgacc 240

atggacggcg gcaccctgcc gcgttccgcg ccgccggcgc cgccagttcc ggttggctgc 300

gctgcccgtc gccgtcccgc gtccccggaa ctgctgcgct gcagccgtcg ccgtcgcccg 360

gccaccgcag agaccggagg cggcgcagcg gccgtagcgc ggcgcaatga gcgcgagcgc 420

aaccgcgtga agctggtgaa cttgggcttc caggcgctgc ggcagcacgt gccgcacggc 480

ggcgccagca agaagctgag caaggtggag acgctgcgct cagccgtgga gtacatccgc 540

gcgctgcagc gcctgctggc cgagcacgac gccgtgcgca acgcgctggc gggagggctg 600

aggccgcagg ccgtgcggcc gtctgcgccc cgcgggccgc cagggaccac cccggtcgcc 660

gcctcgccct cccgcgcttc ttcgtccccg ggccgcgggg gcagctcgga gcccggctcc 720

ccgcgttccg cctactcgtc ggacgacagc ggctgcgaag gcgcgctgag tcctgcggag 780

cgcgagctac tcgacttctc cagctggtta gggggctaca ctagtggcca ccatcaccat 840

caccattaa 849

<210>10

<211>282

<212>PRT

<213> influenza virus and the mankind

<400>10

Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp

1 5 10 15

His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala Pro Phe

20 25 30

Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly Arg Gly Ser

35 40 45

Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala Gly Lys Gln Ile

50 55 60

Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu Ala Leu Lys Met Thr

65 70 75 80

Met Asp Gly Gly Thr Leu Pro Arg Ser Ala Pro Pro Ala Pro Pro Val

85 90 95

Pro Val Gly Cys Ala Ala Arg Arg Arg Pro Ala Ser Pro Glu Leu Leu

100 105 110

Arg Cys Ser Arg Arg Arg Arg Pro Ala Thr Ala Glu Thr Gly Gly Gly

115 120 125

Ala Ala Ala Val Ala Arg Arg Asn Glu Arg Glu Arg Asn Arg Val Lys

130 135 140

Leu Val Asn Leu Gly Phe Gln Ala Leu Arg Gln His Val Pro His Gly

145 150 155 160

Gly Ala Ser Lys Lys Leu Ser Lys Val Glu Thr Leu Arg Ser Ala Val

165 170 175

Glu Tyr Ile Arg Ala Leu Gln Arg Leu Leu Ala Glu His Asp Ala Val

180 185 190

Arg Asn Ala Leu Ala Gly Gly Leu Arg Pro Gln Ala Val Arg Pro Ser

195 200 205

Ala Pro Arg Gly Pro Pro Gly Thr Thr Pro Val Ala Ala Ser Pro Ser

210 215 220

Arg Ala Ser Ser Ser Pro Gly Arg Gly Gly Ser Ser Glu Pro Gly Ser

225 230 235 240

Pro Arg Ser Ala Tyr Ser Ser Asp Asp Ser Gly Cys Glu Gly Ala Leu

245 250 255

Ser Pro Ala Glu Arg Glu Leu Leu Asp Phe Ser Ser Trp Leu Gly Gly

260 265 270

Tyr Thr Ser Gly His His His His His His

275 280

<210>11

<211>193

<212>PRT

The <213> mankind

<400>11

Met Tyr Ser Thr Ala Glu Arg Ser Val Ser Thr Leu Leu Ser Phe Leu

1 5 10 15

Leu Ala Pro Pro Cys Gly Thr Cys Cys Arg Ser Ala Trp Lys Pro Lys

20 25 30

Phe Thr Ser Phe Thr Arg Leu Arg Ser Arg Ser Leu Arg Arg Ala Thr

35 40 45

Ala Ala Ala Pro Pro Pro Val Ser Ala Val Ala Gly Arg Arg Arg Arg

50 55 60

Leu Gln Arg Asn Ser Ser Gly Asp Ala Gly Leu Arg Arg Ala Ala Gln

65 70 75 80

Pro Thr Gly Thr Gly Gly Ala Gly Gly Ala Asp Leu Gly Ser Val Pro

85 90 95

Pro Ser Ile Ala Pro Ala Ser Thr Arg Pro Leu Gln Val Pro Ala Arg

100 105 110

Arg Arg Lys Val Gln Ala Glu Glu Pro Glu Ala Thr Gly Lys Thr Val

115 120 125

Ala Pro Gln Gly Gly Phe Trp His Gly Ala Ala Arg Gln Leu Pro Arg

130 135 140

Ala Arg Val Leu Gly Arg Leu Glu Pro Gly Asp Arg Arg Pro Ser Gly

145 150 155 160

Gly Arg Pro Tyr Ala Pro Gly Ser Val Gly Arg Ser Cys Pro Ala Arg

165 170 175

Ala Ala Gly Leu Ser Gln Val Ser Ala Gly Ala Ala Gln Ala Ala Gly

180 185 190

Phe

<210>12

<211>263

<212>PRT

<213> mice

<400>12

Met Glu Ala His Leu Asp Trp Tyr Gly Val Pro Gly Leu Gln Glu Ala

1 5 10 15

Ser Asp Ala Cys Pro Arg Glu Ser Cys Ser Ser Ala Leu Pro Glu Ala

20 25 30

Arg Glu Gly Ala Asn Val His Phe Pro Pro His Pro Val Pro Arg Glu

35 40 45

His Phe Ser Cys Ala Ala Pro Glu Leu Val Ala Gly Ala Gln Gly Leu

50 55 60

Asn Ala Ser Leu Met Asp Gly Gly Ala Leu Pro Arg Leu Met Pro Thr

65 70 75 80

Ser Ser Gly Val Ala Gly Ala Cys Ala Ala Arg Arg Arg Gln Ala Ser

85 90 95

Pro Glu Leu Leu Arg Cys Ser Arg Arg Arg Arg Ser Gly Ala Thr Glu

100 105 110

Ala Ser Ser Ser Ser Ala Ala Val Ala Arg Arg Asn Glu Arg Glu Arg

115 120 125

Asn Arg Val Lys Leu Val Asn Leu Gly Phe Gln Ala Leu Arg Gln His

130 135 140

Val Pro His Gly Gly Ala Asn Lys Lys Leu Ser Lys Val Glu Thr Leu

145 150 155 160

Arg Ser Ala Val Glu Tyr Ile Arg Ala Leu Gln Arg Leu Leu Ala Glu

165 170 175

His Asp Ala Val Arg Ala Ala Leu Ala Gly Gly Leu Leu Thr Pro Ala

180 185 190

Thr Pro Pro Ser Asp Glu Cys Ala Gln Pro Ser Ala Ser Pro Ala Ser

195 200 205

Ala Ser Leu Ser Cys Ala Ser Thr Ser Pro Ser Pro Asp Arg Leu Gly

210 215 220

Cys Ser Glu Pro Thr Ser Pro Arg Ser Ala Tyr Ser Ser Glu Glu Ser

225 230 235 240

Ser Cys Glu Gly Glu Leu Ser Pro Met Glu Gln Glu Leu Leu Asp Phe

245 250 255

Ser Ser Trp Leu Gly Gly Tyr

260

<210>13

<211>1051

<212>DNA

<213> mice

<400>13

gcccggagca tggaagcacg tcagctaggc catgaactgc acccgggagg ggtgggggtg 60

gaagcgcacg gtgtcagctt tgcagaatgt gtacaccaag gggagggcga ggcgaaggaa 120

ggagggcgta agaaaggagg cggtggcggg gcggaggaga ttatctatac tttttaaaaa 180

aaaggagcct cttagccgcg taaaggagac ttggggagcg cctgacagca cgcgcgggac 240

acgagagtac cacgcttccc tactcttttc agaccttgac tggtacgggg tcccaggact 300

gcaggaggcc agcgacgcgt gccctaggga gtcctgcagc agtgccctgc ctgaggcccg 360

tgaaggtgca aacgtccact tcccaccgca cccggttcct cgcgagcact tttcctgtgc 420

cgcaccagaa ctcgtagcag gggcccaggg gctgaatgca agcttgatgg acggcggcgc 480

gctgcccaga ctcatgccca cctcgtctgg agtcgctgga gcctgcgctg ctcggcggag 540

acaagcgtct ccggaattgc tgcgctgcag ccggcggcgg cgatctggag caaccgaggc 600

cagcagcagc tcggcgtccg tggcacgccg caatgagcgc gagcgcaacc gcgtaaagct 660

ggtaaacttg ggcttccagg cgctgcggca gcacgtgccg cacggcggcg ccaacaagaa 720

gctgagtaag gtggagacgc tgcgctccgc ggtagagtac attcgtgcgc tgcagcggct 780

gctcgcagag cacgacacgg tgcggccggn gctcgctggg gggctgttaa cacccgctac 840

tccgccgtcc gatgagtgca cgcagccctc tgcctcccct gccagcgggt ctctgtcctg 900

cgcctctacg tctccgtccc ggaccctggg ctgctctgag cctacctccc cgcgctccgc 960

ctactcgtcg gaggaaagca gctgcgaggg agagctaagc ccgatggagc aggagctgct 1020

tgacttttcc agttggttag ggggctactg a 1051

<210>14

<211>260

<212>PRT

<213> rat

<400>14

Met Glu Sar His Phe Asn Trp Tyr Gly Val Pro Arg Leu Gln Lys Ala

1 5 10 15

Ser Asp Ala Cys Pro Arg Glu Ser Cys Ser Ser Ala Leu Pro Glu Ala

20 25 30

Arg Glu Gly Ala Asn Val His Phe Pro Pro His Pro Val Pro Arg Glu

35 40 45

His Phe Ser Cys Gly Ala Pro Lys Pro Val Ala Gly Ala Pro Ala Leu

50 55 60

Asn Ala Ser Leu Met Asp Gly Gly Ala Leu Pro Arg Leu Val Pro Thr

65 70 75 80

Ser Ser Gly Val Ala Gly Ala Cys Thr Ala Arg Arg Arg Pro Pro Ser

85 90 95

Pro Glu Leu Leu Arg Cys Ser Arg Arg Arg Arg Ser Gly Ala Thr Glu

100 105 110

Ala Ser Ser Ser Ser Ala Ala Val Ala Arg Arg Asn Glu Arg Glu Arg

115 120 125

Asn Arg Val Lys Leu Val Asn Leu Gly Phe Gln Ala Leu Arg Gln His

130 135 140

Val Pro His Gly Gly Ala Asn Lys Lys Leu Ser Lys Val Glu Thr Leu

145 150 155 160

Arg Ser Ala Val Glu Tyr Ile Arg Ala Leu Gln Arg Leu Leu Ala Glu

165 170 175

His Asp Ala Val Arg Ala Ala Leu Ser Gly Gly Leu Leu Thr Pro Ala

180 185 190

Thr Arg Pro Ser Asp Val Cys Thr Gln Pro Ser Ala Ser Pro Ala Ser

195 200 205

Ala Ser Leu Ser Cys Thr Ser Thr Ser Pro Asp Arg Leu Gly Cys Ser

210 215 220

Glu Pro Ala Ser Pro Arg Ser Ala Tyr Ser Ser Glu Asp Ser Ser Cys

225 230 235 240

Glu Gly Glu Thr Tyr Pro Met Gly Gln Met Phe Asp Phe Ser Asn Trp

245 250 255

Leu Gly Gly Tyr

260

<210>15

<211>1526

<212>DNA

<213> rat

<400>15

ttcacccggc tgcaagcgct aggtgtacgg agacctggca gctcttgggg cttaaggact 60

gagcrccaga gccggtggag gttcctgtgg agtacattcg gaccctctca cagcccccga 120

gagtgcggga cgtgcggagc gcagttcggg atctgcactc gaggacttgt cgaggacgca 180

ttaagctaag catctgctcg gagcatggaa tcgcacttta actggtacgg ggtcccaagg 240

ctccagaagg ctagcgacgc gtgccctagg gaatcctgca gcagtgccct gcctgaggcc 300

cgtgaaggtg cgaacgtcca cttcccaccg cacccggttc ctcgcgagca cttttcctgt 360

ggcgcaccga aacccgtagc gggggccccg gcgctgaatg caagcttgat ggacggcggc 420

gcgctgccca gactcgtgcc cacctcgtct ggagtcgctg gagcctgcac tgctcggcgg 480

agacccccgt ccccggaact gcttcgctgc agccgacggc ggcgatcggg agcaaccgag 540

gccagcagca gctcggcggc cgtggcacgc cgcaatgagc gtgagcgcaa ccgcgtaaag 600

ctggtaaact tgggcttcca ggcgctgcgg cagcacgtgc cgcacggcgg cgccaacaag 660

aagctgagta aggtggagac gctgcgctcc gcggtagagt acatccgtgc gctgcagcgg 720

ctgctagcag agcacgacgc ggtgcgtgct gcgctctctg ggggtctatt aacacccgct 780

actcggccgt ccgatgtgtg cacgcagccc tccgcctccc ctgccagcgc gtctctgtcc 840

tgcacctcta catccccaga ccgcctaggc tgctccgagc ctgcctctcc gcgctccgcc 900

tactcgtcgg aggacagcag ctgcgaggga gagacttacc cgatggggca gatgtttgac 960

ttttccaatt ggttaggggg ctactgagca ccccacaccc ctaagctgcg tccctgggtg 1020

tcccctggtg gacctacctg cgtttcttgc ccaggaaacc tgggcccatg ccttacccat 1080

gctgtctagt gcagcctgac caaatgccaa gtactgacct ctgctcggcc tccacgccgc 1140

ggaatgacat cttccatctc ccagtccttg ccgaaccagg acttggaaat ttctcaggag 1200

aaagaatttt acaatgacaa tctgcttttt atcaattaac ttgaactgct ggaggactct 1260

gctgaaaata tgaagaatta tttttataca aaggatcctt aagcttggag cacaataaag 1320

atgacctctg tctctcaccc ccactgtcta gaactttcca acctggccaa agtgtggacg 1380

ggtcgggccc tgagggcaag atgcctggct gcacccttct tcctcttccg aagcctatcc 1440

tgacgctgat gtttggccag tgtgggaacc ctgctattgc aaagtgtact attctataaa 1500

agttgttttt cattggaaag gaattc 1526

<210>16

<211>10

<212>PRT

The <213> mankind

<400>16

Lys Leu Val Asn Leu Gly Phe Gln Ala Leu

1 5 10

<210>17

<211>9

<212>PRT

The <213> mankind

<400>17

Glu Leu Leu Asp Phe Ser Ser Trp Leu

1 5

<210>18

<211>9

<212>PRT

The <213> mankind

<400>18

Arg Leu Leu Ala Glu His Asp Ala Val

1 5

<210>19

<211>9

<212>PRT

The <213> mankind

<400>19

Lys Leu Val Asn Leu Gly Phe Gln Ala

1 5

<210>20

<211>9

<212>PRT

The <213> mankind

<400>20

Glu Tyr Ile Arg Ala Leu Gln Arg Leu

1 5

<210>21

<211>10

<212>PRT

The <213> mankind

<400>21

Glu Tyr Ile Arg Ala Leu Gln Arg Leu Leu

1 5 10

<210>22

<211>10

<212>PRT

The <213> mankind

<400>22

Ala Val Arg Asn Ala Leu Ala Gly Gly Leu

1 5 10

<210>23

<211>10

<212>PRT

The <213> mankind

<400>23

Ser Glu Pro Gly Ser Pro Arg Ser Ala Tyr

1 5 10

<210>24

<211>10

<212>PRT

The <213> mankind

<400>24

Val Glu Thr Leu Arg Ser Ala Val Glu Tyr

1 5 10

<210>25

<211>9

<212>PRT

The <213> mankind

<400>25

Ile Arg Ala Leu Gln Arg Leu Leu Ala

1 5

<210>26

<211>9

<212>PRT

The <213> mankind

<400>26

Leu Arg Pro Gln Ala Val Arg Pro Ser

1 5

<210>27

<211>9

<212>PRT

The <213> mankind

<400>27

Leu Arg Gln His Val Pro His Gly Gly

1 5

<210>28

<211>9

<212>PRT

The <213> mankind

<400>28

Leu Gly Phe Gln Ala Leu Arg Gln His

1 5

<210>29

<211>9

<212>PRT

<213>Human

<400>29

Val Arg Asn Ala Leu Ala Gly Gly Leu

1 5

<210>30

<211>9

<212>PRT

The <213> mankind

<400>30

Tyr Ile Arg Ala Leu Gln Arg Leu Leu

1 5

<210>31

<211>9

<212>PRT

The <213> mankind

<400>31

Leu Val Asn Leu Gly Phe Gln Ala Leu

1 5

<210>32

<211>9

<212>PRT

The <213> mankind

<400>32

Val Glu Tyr Ile Arg Ala Leu Gln Arg

1 5

<210>33

<211>9

<212>PRT

The <213> mankind

<400>33

Leu Leu Arg Cys Ser Arg Arg Arg Arg

1 5

Claims (4)

1. The purposes of the polypeptide of the aminoacid sequence that 1.SEQ ID NO:2 represents in the immunogenic composition for the preparation of prevention or treatment cancer, described immunogenic composition comprises: polypeptide, TH1 induction type adjuvant and the pharmaceutically acceptable carrier of the aminoacid sequence that SEQ ID NO:2 represents.
2. 2. the purposes of claim 1, wherein said TH-1 induction type adjuvant is selected from the mixture, CpG ODN of following adjuvant: 3D-MPL, QS21, QS21 and cholesterol or the mixture of adjuvant described in two or more.
3. 3. the purposes of the polynucleotide of separation in the reagent for the preparation of cancer diagnosis, the polypeptide of any one in described polynucleotide encoding claim 1-2.
4. 4. the purposes of immune specificity antibody in the reagent for the preparation of cancer diagnosis, described antibody has immune specificity for the polypeptide of claim 1, and wherein said antibody produces by the immunogenic described polypeptide as tumor associated antigen.

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