CN1948333B

CN1948333B - New hPEBP4 protein source HLA-A2 limiting epi-polypeptide and its application

Info

Publication number: CN1948333B
Application number: CN2005100305320A
Authority: CN
Inventors: 李楠; 孙伟红; 王晓健; 曹雪涛
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2005-10-14
Filing date: 2005-10-14
Publication date: 2010-12-08
Anticipated expiration: 2025-10-14
Also published as: CN1948333A

Abstract

The present invention relates to a HLA-A2 restriction epitope polypeptide formed from hPEBP4 protein and said epitope and its related recombinant protein, coding nucleotide sequence, antigen presentating cell, composition and application for preventing and curing hPEBP4 tumor.

Description

A kind of HLA-A2 restriction epi polypeptide and application thereof of new hPEBP4 dietary protein origin

Technical field

The present invention relates to biology and medical field, relate more specifically to a kind of HLA-A2 restriction epi polypeptide of hPEBP4 dietary protein origin, and this epi-position and relevant recombinant protein thereof, coding nucleotide sequence, antigen presenting cell, composition and at the purposes of specific immunity effector cell in expressing hPEBP4 oncotherapy and prevention of this epi-position.

Background technology

HPEBP4 (SEQ ID NO:12), hPEBP4 is former to be called " hPEBP5 ", and its nucleotide sequence and amino acid are seen Chinese patent application CN02136556.3.It is to support acquisition marrow stromal cell (Bone marrow stromal cell from external former being commissioned to train of normal adult medullary cell, BMSC) and make up BMSC cDNA library, utilize the means of cDNA library large scale sequencing, a kind of novel full-length cdna that from BMSC cDNA library, is separated to, belong to phosphotidylethanolabinding binding protein (PEBP) family, login (sequence number is AY037148) in the GenBank/EMBL database.HPEBP4 encoding histone 227 amino acid (SEQ ID NO:13).Its aminoacid sequence is that typical phosphatidylethanolamine is in conjunction with conservative region (PBP) in the 75-195 position.

Synthetic peptide vaccine is in recent years along with molecular biology and immunologic progress and a kind of new vaccine that grows up, can induce body to produce antigen-specific immune responses, and its side effect is slight, security good, be a new direction of present vaccine research, widespread use and antitumor and antiviral immunity treatment.At present there has been multiple vaccine to enter clinical study or listing based on epitope polypeptide.

Cellular immunization plays a crucial role in antitumor and antiviral immunity.The antigen of T cell recognition is and the MHC-I class or the II quasi-molecule bonded peptide of cell surface that its length is about 8-12 amino acid.And then discern and MHC-I quasi-molecule bonded endogenous peptide as the main effects cell CTL (cytotoxic T cell, Cytotoxic TLymphocytes) of killing tumor cell.Show that on evidence synthetic Toplink directly combines with the MHC-I quasi-molecule, and do not need processing, the processing of APC, it and natural endogenous peptide have equally valid aspect activating immune system.

Polypeptide vaccine has become a New Policy of present anti-malignant tumor, also is to study maximum tumor therapeutic vaccines at present.Study more having: (1) gp100: (sequence is: KTWGQYWQV), (sequence is G9209: ITDQVPPFSV) CTL that all can inducing antigen-specific to derive from the peptide G9154 of gp100.With sensitization from HLA-A2 ⁺The peripheral blood lymphocyte (PBL) of melanoma patients, can obviously strengthen the ability of its inducing specific CTL: inductive CTL can discern the gp100 of unmodified; (2) CEA: adopt and above-mentioned similar methods, people such as Zaremba studies show that: the peptide CAP1-6D of CEA can not only be at the special CTL of external sensitization CEA, its render a service for the 100-1000 of CAP1 doubly, also can induce the special CTL of CEA (and CAP1 can not) in vivo; And the CTL of institute's sensitization can discern CAP1 equally.What is more important, these CTL can dissolve the human tumor that homologous is expressed CEA; (3) MAGE-2:MAGE-2 extensively is present in kinds of tumors (except that testis) such as melanoma, laryngocarcinoma, lung cancer, sarcoma, is not present in healthy tissues.Found that in MAGE-2 three sections polypeptide can form stable compound down at 37 ℃ with the MHC-I quasi-molecule, wherein had at least two energy to be processed, present, become the new candidate of polypeptide vaccine research by HLA-A*0201; (4) P21 ^WAFI: with P21 ^WAFIPeptide and endogenousization peptide merge and can suppress growth of tumor; (5) HER2/neu: the peptide that derives from HER2/neu also can be induced special CTL, and can suppress growth of tumor at the specific CTL of external energy sensitization in the mouse body.Adopt the HPV polypeptide vaccine also to enter clinical study in addition.

HLA-A2 is a kind of a kind of MHC-I quasi-molecule that has higher distribution in population of China, positive rate is between 40-60%, account for the first place of each subgroup of MHC-I quasi-molecule, because HLA-A*0201 is the highest site of crowd's frequency of occurrences, motif is formed clear and definite, therefore is associated molecule first-selected in the vaccine design.Along with dark people to MHC-I quasi-molecule and peptide epitopes interaction of molecules understanding, scientists has been set up comparatively sophisticated epi-position authenticate technology route, key step is as follows: (1) is according to epi-position and the interactional characteristics of MHC-I quasi-molecule, prediction MHC-I quasi-molecule restricted CTL epitope, and utilize computer molecular simulateo that the prediction epi-position is further screened; (2) measure epi-position and MHC-I quasi-molecule bonding force; (3) utilize analysis of cell in vitro poison or immune lotus knurl transgenic mice evaluation peptide epitopes can induce CTL, seek the optimal vigor epi-position.Based on this technological line, existing multiple HLA-A*0201 restricted CTL epitope identified, what have has shown better curative effect clinical.

The T2 cell is to be used to one of instrument cell of measuring epi-position and HLA-A*0201 molecule bonding force, it is the HLA-A*0201 type cell strain of a strain antigen presentation transporter defective, this cell surface is only expressed the HLA-A*0201 molecule that does not contain the endogenous antigen molecule, thereby can utilize the combination degree of itself and purpose peptide to measure avidity between HLA-A0201 molecule and purpose epi-position.

Still lack at present and effectively prevent and treat antineoplastic therapeutic vaccine, especially safe synthetic peptide vaccine, so this area presses for safe, synthetic peptide vaccine with high immunogenicity that new can be used for of exploitation prevented and treated tumour.

Summary of the invention

Purpose of the present invention just provides a kind of safe, synthetic peptide and application thereof with high immunogenicity.

In a first aspect of the present invention, a kind of hPEBP4 is provided the HLA-A2 restriction epi polypeptide in source, it is characterized in that described polypeptide has the inducing cytotoxic T cell killing activity, and be selected from down group:

(a) comprise the polypeptide of the aminoacid sequence in the following general formula I:

X _aa1-TLFCQGLEV-X _aa2 (I)

In the formula, X _Aa1And X _Aa2Respectively be 0,1,2 or 3 optional amino acid;

(b) the general formula aminoacid sequence is formed through replacement, disappearance or the interpolation of 1-4 amino-acid residue, and have inducing cytotoxic T cell killing activity function by (a) deutero-HLA-A2 restriction epi polypeptide.

More preferably, described amino acid is selected from down group: Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val.

More preferably, described X _Aa1Be do not exist, D, ED or DED; And X _Aa2Be do not exist, F, FY or FYP.

Preferably, described aminoacid sequence is through one or more amino acid whose replacements, disappearance or interpolation.

In an embodiment of the invention, described polypeptide is TLFCQGLEV (SEQ ID NO:1).

A second aspect of the present invention relates to a kind of recombinant protein, and described recombinant protein contains the HLA-A2 restriction epi polypeptide in foregoing hPEBP4 source.

A third aspect of the present invention relates to a kind of isolating nucleic acid, the HLA-A2 restriction epi polypeptide in the foregoing hPEBP4 of described nucleic acid encoding source.

A fourth aspect of the present invention relates to a kind of antigen presenting cell, the HLA-A2 restriction epi polypeptide sensitization that described antigen presenting cell is originated by foregoing hPEBP4.

Preferably, described antigen presenting cell is selected from down group: dendritic cell, scavenger cell, B cell, inoblast, endotheliocyte.

A fifth aspect of the present invention relates to a kind of composition, and described composition contains: (a) the HLA-A2 restriction epi polypeptide or the foregoing antigen presenting cell in the foregoing hPEBP4 of 0.001-99.99wt% source; (b) acceptable carrier, thinner or vehicle.

Preferably, described composition is a pharmaceutical composition, and described carrier, thinner or vehicle are pharmaceutically acceptable carrier, thinner or vehicle.

A sixth aspect of the present invention relates to the HLA-A2 restriction epi polypeptide or the purposes of antigen presenting cell in the medicine of preparation prevention and treatment tumour in a kind of foregoing hPEBP4 source.

In a seventh aspect of the present invention, the purposes of the HLA-A2 restriction epi polypeptide in hPEBP4 of the present invention source is provided, it is used to prepare the recombinant protein (as fusion rotein) that comprises this restriction epi polypeptide, or is used to prepare the antigen presenting cell of sensitization.

Description of drawings

Fig. 1: induce HLA-A2.1/K in dendritic cell (DC) body of P40-48 peptide sensitization ^bThe killing activity of transgenic mice cytotoxic T cell.Wherein the effector cell of A figure is from inductive cytotoxic T cell in the simple usefulness homology DC body; The effector cell of B figure is from inductive cytotoxic T cell in the DC body of P40-48 peptide sensitization.

Fig. 2: induce HLA-A2.1/K in dendritic cell (DC) body of P40-48 peptide sensitization ^bTransgenic mice cytotoxic T cell IFN-γ secretes situation.Wherein the effector cell of A figure is from inductive cytotoxic T cell in the simple usefulness homology DC body; The effector cell of B figure is from inductive cytotoxic T cell in the DC body of P40-48 peptide sensitization.

Embodiment

The inventor finds the hPEBP4 high expression level in some tumour cell through extensive and deep research, can turn up by inhibition Ras/Raf/MEK/ERK approach, JNK activation and PE to suppress TNF α inductive apoptosis.Therefore, utilize the special antigen peptide of hPEBP4, can induce body to produce specific CTL, the tumour cell that kills and wounds high expression level hPEBP4.In view of the above, the inventor is carrying out hPEBP4 on the basis of computer simulation analysis, many the special antigen peptide of hPEBP4 that might combine and induce body to produce CTL have optionally been synthesized with HLA-A*0201, by the T2 cell in conjunction with experiment, filter out the epitope peptide that has strong affinity with HLA-A*0201, and its immunogenicity estimated, find that it not only can be at HLA-A*0201/K ^bInduce specific, the restrictive cytotoxic T cell of HLA-A*0201 in transgenic mice and the healthy human peripheral blood, and be one by cell process naturally, the immunogenic polypeptide of submission.Finished the present invention on this basis.

Particularly, the inventor studies show that, the aminoacid sequence of hPEBP4 is that typical phosphatidylethanolamine is in conjunction with conservative region (PBP) in the 75-195 position.HPEBP4 can turn up by inhibition Ras/Raf/MEK/ERK approach, JNK activation and PE and suppress TNF α inductive apoptosis, and these functions mediate in conjunction with conservative region (PBP) by its PE.

RT-PCR shows, hPEBP4 is as anti-apoptosis molecule high expression level in tumour cells such as prostate cancer PC-3, ovarian cancer CaoV-3, mammary cancer MCF-7, and most of solid tumor cells such as LoVo cell in the A549 cell in lung cancer source and colorectal carcinoma source do not see Table and reach.In the clinical tumor sample, hPEBP4 albumen high expression level and only is expressed in 4% the normal galactophore tissue in the breast cancer tumour tissue more than 50%.In addition, experimental result shows that also the hPEBP4 down-regulated expression has strengthened tumour cells such as mammary cancer, the ovarian cancer susceptibility apoptosis-induced to TNF α, shows that hPEBP4 is tumour potential action target spots such as treatment mammary cancer.

Therefore,, body is carried out immunity, body is produced at the proteic specific immune response of hPEBP4, thereby make body can suppress, remove tumour cell effectively, for the prevention and the treatment of related neoplasms provides measure if be target with hPEBP4.

In view of the above, the inventor is carrying out hPEBP4 on the basis of computer simulation analysis, many the special antigen peptide of hPEBP4 that might combine and induce body to produce CTL have optionally been synthesized with HLA-A*0201, by the T2 peptide in conjunction with experiment, filter out the epitope peptide that has strong affinity with HLA-A*0201, and its immunogenicity estimated, find that it not only can be at HLA-A*0201/K ^bInduce specific, the restrictive cytotoxic T cell of HLA-A*0201 in transgenic mice and the healthy human peripheral blood, and be one by cell process naturally, the immunogenic polypeptide of submission.

The evaluation of the restrictive cytotoxic T cell epitope peptide of HLA-A2 in tumor associated antigen hPEBP4 source has important meaning to the development of its tumor vaccine and treatment preparation.

As used herein, " polypeptide of the present invention ", " specific polypeptide ", " HLA-A2 restriction epi polypeptide " are used interchangeably, and refer to the polypeptide shown in the aminoacid sequence P40-48 (SEQ ID NO:1).In addition, also comprise having and P40-48 (SEQ ID NO:1) variant form identical function, SEQ ID NO:1 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-20, preferably 1-10, more preferably 1-5,1-3 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the reactive derivative of P40-48.

A kind of polypeptide of preferably deriving is 1 or 2 or 3 or 4 amino acid (as D, ED, DED or LDED) of 36-39 position in the upstream of SEQ ID NO:1 is added corresponding to hPEBP4, and/or in the downstream of SEQ ID NO:1 is added corresponding to hPEBP4 1 or 2 or 3 or 4 amino acid (as F, FY, FYP or FYPE) of 49-52 position.

Polypeptide of the present invention can be used the ordinary method synthetic, also available recombination method production.

Polypeptide of the present invention also can be used for the albumen coupling with the BSA equimolecular quantity, thereby forms the polypeptide conjugate.Usually, described conjugate is made of polypeptide, linking agent and BSA, the preferred glutaraldehyde of wherein said linking agent, EDAC.

In addition, polypeptide of the present invention and conjugate also can be used for preparing curative pharmaceutical composition or preventative and curative vaccine composition.

Therefore, on the other hand, the present invention also provides a kind of composition, and it contains polypeptide of the present invention, conjugate or its composition of (a) safe and effective amount; And (b) pharmaceutically acceptable carrier or vehicle.The quantity of polypeptide of the present invention is generally 10 micrograms-100 milligram/agent, preferably is 100-1000 microgram/agent.

Term used herein " significant quantity " refers to the therapeutical agent treatment, alleviates or prevent the amount of target disease or situation, or shows the amount of detectable treatment or preventive effect.Depend on the nature and extent of the build of this object and healthy state, illness and the therapeutical agent selecting to give and/or the combination of therapeutical agent for the accurate significant quantity of a certain object.Therefore, specifying accurately in advance, significant quantity is useless.Yet, for certain given situation, can determine this significant quantity with normal experiment, the clinicist can judge.

For the purposes of the present invention, effective dosage is for giving individual about 0.01 mg/kg to 50 mg/kg, the preferably polypeptide of the present invention of 0.05 mg/kg to 10 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

Pharmaceutical composition also can contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration.This term refers to like this some medicament carriers: they itself do not induce generation to accepting the individual deleterious antibody of said composition, and do not have undue toxicity after the administration.These carriers are well known to those of ordinary skill in the art.(Mack Pub.Co. can find discussing fully about pharmaceutically acceptable vehicle in N.J.1991) at Remington ' s Pharmaceutical Sciences.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol, adjuvant and combination thereof.

Acceptable carrier can contain liquid on the therapeutic composition Chinese materia medica, as water, salt solution, glycerine and ethanol.In addition, also may there be complementary material in these carriers, as wetting agent or emulsifying agent, pH buffer substance etc.In addition, can also contain immunological adjuvant in the immune composition.

Usually, therapeutic composition can be made injectable agent, for example liquor or suspension; Also can be made into before injection, be fit to allocate in solution or the suspension, the solid form of liquid vehicle.

In case be made into composition of the present invention, can directly give object with it.The object of waiting to prevent or treating can be an animal; Especially people.

Treatment or the prophylactic medicament (comprising vaccine) that contains polypeptide of the present invention of the present invention can oral administration, mode such as subcutaneous, intracutaneous, intravenous injection uses.The therapeutic dose scheme can be single agent scheme or multi-agent scheme.

Major advantage of the present invention is: epitope peptide of the present invention is the restrictive cytotoxic T cell epitope peptide of hPEBP4 dietary protein origin, HLA-A2, can cause immunne response safely and effectively at tumour cell, then not only to the tumor invasion Study on Mechanism, and to the development of tumor therapeutic vaccine and treatment preparation important meaning is arranged all.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment

The screening of embodiment 1HLA-A*0201 high-affinity peptide

Present embodiment is selected epitope peptide with the HLA-A*0201 high-affinity by peptide in conjunction with testing sieve.

Testing sequence

At first collect T2 cell (a kind of cell that lacks the antigen working ability, buy from ATCC:CRL-1991), after giving a baby a bath on the third day after its birth time with serum-free 1640, accent cell concn to 2 * 10 ⁵/ ml is laid in 24 orifice plates 0.5ml/ hole.Again with candidate's polypeptide of 50 μ M, the β2Wei Qiudanbai of 2.5 μ g/ml is in 37 ℃, 5%CO ₂Hatch 18h in the incubator altogether.The cell of hatching is given a baby a bath on the third day after its birth time with ice PBS, and (UK), ice bath 45 minutes, PBS are washed the back and detected average fluorescent strength with flow cytometer for Sterotec Ltd, Oxford to add the specific mAb BB7.2 of HLA-A2 of FITC mark.As positive control, the simple T2 cell that not adding peptide stimulates contrasts as a setting with positive known peptide CEA HLA-A2 restriction epi polypeptide CAP-1 (SEQ ID NO:10, sequence is YLSGANLNL).

Detection method

The situation that combines of immunofluorescence technique detection of peptides and HLA-A*0201 molecule, being based on exogenous polypeptid increases with the expression amount that combining of T2 cell surface MHC-I quasi-molecule can make its surperficial MHC-I quasi-molecule, both are in conjunction with firm more, expression amount that then can detected MHC-I quasi-molecule is many more, with the average fluorescent strength is to detect index.The result with fluorescence coefficient (FI) as measurement index.The FI of polypeptide＞1 is considered to the epi-position of high-affinity.

Test-results

The T2-HLA-A*0201 bonded avidity result of the hPEBP4 dietary protein origin polypeptide that immunofluorescence technique records is as shown in table 1.The inventor filters out the epi-position P40-48 of HLA-A2 high-affinity from several hPEBP4 dietary protein origin polypeptide, its sequence is TLFCQGLEV (SEQ ID NO:1).

The T2-HLA-A*0201 bonded avidity of table 1hPEBP4 dietary protein origin polypeptide

High-affinity: FI＞1

Embodiment 2 expresses the preparation of the adenovirus (pAdPEBP4) of hPEBP4

Operate according to the Adeasy of Stratagene company adenovirus system test kit specification sheets.Concise and to the point step is:

With the plasmid that comprises human hPEBP 4 cDNA that obtained among the Chinese patent application CN02136556.3 embodiment 1 is template, cut through SalI-Not I enzyme, recombinate according to a conventional method with plasmid pShuttle-CMV (Stratagene company) again and be converted into the competence e. coli bl21, the picking positive colony is identified back purifying and order-checking (ABI company, BigDye Terminator test kit).With the human hPEBP 4-pShuttle-CMV carrier of correct sequence after Pme I linearizing with the common transformed into escherichia coli BJ5183 (Stratagene company) of pAdeasy adenovirus skeleton plasmid (Stratagene company).Positive colony is cut evaluation with the PacI enzyme, the capable 0.8% agarose gel electrophoresis analysis of product.Verified, having recombinated has produced the adenovirus carrier that comprises the human hPEBP 4 encoding sequence.

This adenovirus carrier utilizes Lipofectamine transfection reagent (Invitrogen) the transfection AD293 of company cell (Stratagene company) after the PacI linearizing, treat after 7-10 days that cell becomes round fully, collecting cell, multigelation obtains viral supernatant, carry out the purifying of a large amount of viruses by the CsCl gradient centrifugation, thereby obtain to express the recombinant adenovirus (pAdhPEBP4) of hPEBP4, virus titer is 10 * 10 ¹³

Embodiment 3HLA-A2.1/K ^bInducing of the restricted polypeptid specificity cytotoxic T cell of HLA-A2 of originating at hPEBP4 in the transgenic mice body

Effector cell's preparation

Prepare HLA-A2.1/K according to a conventional method ^bThe dendritic cell of transgenic mice derived from bone marrow (DC).Collection is cultured to the 7th day DC, transfers cell concn to 1 * 10 ⁶Individual cell/ml adds P40-48 (final concentration 10 μ M/ml) and β2Wei Qiudanbai (final concentration 3 μ g/ml), and 37 ℃, 5%CO ₂Hatch 3h in the incubator.Collect the DC of peptide sensitization, give a baby a bath on the third day after its birth time, transfer cell concn to 1 * 10 with PBS ⁶/ 0.2ml immune mouse.Every mouse peritoneal injection 1 * 10 ⁶Individual cell/0.2ml, immunity is three times altogether, at interval a week.Back 7 days of last immunity, mouse spleen is won in aseptic technique, and lysed erythrocyte is made single cell suspension.With splenocyte suspension (5 * 10 ⁶/ ml) autologous dendritic cell through irradiation with peptide sensitization places RPMI 1640 perfect mediums to cultivate with 10: 1 ratios.Cultivate after 6 days, collect the effector cell,

Detection method

(1) the active detection of specific killing

Present embodiment has adopted 4 hours of standard ⁵¹Cr release test detection specificity killing activity.

Use load P 40-48, SSp-1 (irrelevant contrast respectively, sequence is: T2 (positive control) that the recombinant adenovirus (pAdPEBP4) of T2 cell RLNEVAKNL (SEQ ID NO:11)), expression hPEBP4 infects and unloaded T2 cell add as target cell ⁵¹Cr (100 μ Ci/10 ⁶Individual cell), put in 37 ℃ of water-baths mark 90 minutes, the 15 minutes light mixings in every interval are once washed 3 times, thoroughly the remaining Na of flush away ₂ ⁵¹CrO ₄, it is 1 * 10 that the target cell that mark is good is adjusted cell concn with perfect medium ⁵Individual cell/ml adds 96 hole circle base plates, every hole 100 μ l., hatched 4 hours for 37 ℃ than adding the effector cell by 50: 1,25: 1,12.5: 1 three different targets of imitating, collect each 100 μ l of each hole supernatant, detect the cpm value with the γ calculating instrument.It is that independent target cell adds 100 μ l 1%SDS that each hole is organized in maximum release; Spontaneous release aperture is that independent target cell adds 100 μ l perfect mediums.

(2) detection of IFN-γ in the peptide specific CTL born of the same parents

Add the corresponding peptide of 20 μ M among the inductive effector cell of institute, after 48 hours, dyeing in the conventional born of the same parents.

Test-results

The result shows that the dendritic cell of the restricted polypeptide P40-48 of the HLA-A2 sensitization in hPEBP4 source can significantly be induced HLA-A2.1/K ^bTransgenic mice produces restricted polypeptid specificity cytotoxic T cell and killing activity (see figure 1), and can stimulate IFN-γ to produce (see figure 2).

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Second Military Medical University, PLA

＜120〉a kind of HLA-A2 restriction epi polypeptide and application thereof of new hPEBP4 dietary protein origin

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Ile?Gln?Gly?Gln?Glu?Leu?Ser?Ala?Tyr?Gln?Ala?Pro?Ser?Pro?Pro?Ala

130 135 140

cac?agt?ggc?ttc?cat?cgc?tac?cag?ttc?ttt?gtc?tat?ctt?cag?gaa?gga 542

His?Ser?Gly?Phe?His?Arg?Tyr?Gln?Phe?Phe?Val?Tyr?Leu?Gln?Glu?Gly

145 150 155

aaa?gtc?atc?tct?ctc?ctt?ccc?aag?gaa?aac?aaa?act?cga?ggc?tct?tgg 590

Lys?Val?Ile?Ser?Leu?Leu?Pro?Lys?Glu?Asn?Lys?Thr?Arg?Gly?Ser?Trp

160 165 170 175

aaa?atg?gac?aga?ttt?ctg?aac?cgt?ttc?cac?ctg?ggc?gaa?cct?gaa?gca 638

Lys?Met?Asp?Arg?Phe?Leu?Asn?Arg?Phe?His?Leu?Gly?Glu?Pro?Glu?Ala

180 185 190

agc?acc?cag?ttc?atg?acc?cag?aac?tac?cag?gac?tca?cca?acc?ctc?cag 686

Ser?Thr?Gln?Phe?Met?Thr?Gln?Asn?Tyr?Gln?Asp?Ser?Pro?Thr?Leu?Gln

195 200 205

gct?ccc?aga?gaa?agg?gcc?agc?gag?ccc?aag?cac?aaa?aac?cag?gcg?gag 734

Ala?Pro?Arg?Glu?Arg?Ala?Ser?Glu?Pro?Lys?His?Lys?Asn?Gln?Ala?Glu

210 215 220

ata?gct?gcc?tgc?tagatagccg?gctttgccat?ccgggcatgt?ggccacactg 786

Ile?Ala?Ala?Cys

225

cccaccaccg?acgatgtggg?tatggaaccc?cctctggata?cagaacccct?tcttttccaa 846

ataaaaaaaa?aatcatccag?gaaaaaaa 874

<210>13

<211>227

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>13

Met?Gly?Trp?Thr?Met?Arg?Leu?Val?Thr?Ala?Ala?Leu?Leu?Leu?Gly?Leu

1 5 10 15

Met?Met?Val?Val?Thr?Gly?Asp?Glu?Asp?Glu?Asn?Ser?Pro?Cys?Ala?His

20 25 30

Glu?Ala?Leu?Leu?Asp?Glu?Asp?Thr?Leu?Phe?Cys?Gln?Gly?Leu?Glu?Val

35 40 45

Phe?Tyr?Pro?Glu?Leu?Gly?Asn?Ile?Gly?Cys?Lys?Val?Val?Pro?Asp?Cys

50 55 60

Asn?Asn?Tyr?Arg?Gln?Lys?Ile?Thr?Ser?Trp?Met?Glu?Pro?Ile?Val?Lys

65 70 75 80

Phe?Pro?Gly?Ala?Val?Asp?Gly?Ala?Thr?Tyr?Ile?Leu?Val?Met?Val?Asp

85 90 95

Pro?Asp?Ala?Pro?Ser?Arg?Ala?Glu?Pro?Arg?Gln?Arg?Phe?Trp?Arg?His

100 105 110

Trp?Leu?Val?Thr?Asp?Ile?Lys?Gly?Ala?Asp?Leu?Lys?Lys?Gly?Lys?Ile

115 120 125

Gln?Gly?Gln?Glu?Leu?Ser?Ala?Tyr?Gln?Ala?Pro?Ser?Pro?Pro?Ala?His

130 135 140

Ser?Gly?Phe?His?Arg?Tyr?Gln?Phe?Phe?Val?Tyr?Leu?Gln?Glu?Gly?Lys

145 150 155 160

Val?Ile?Ser?Leu?Leu?Pro?Lys?Glu?Asn?Lys?Thr?Arg?Gly?Ser?Trp?Lys

165 170 175

Met?Asp?Arg?Phe?Leu?Asn?Arg?Phe?His?Leu?Gly?Glu?Pro?Glu?Ala?Ser

180 185 190

Thr?Gln?Phe?Met?Thr?Gln?Asn?Tyr?Gln?Asp?Ser?Pro?Thr?Leu?Gln?Ala

195 200 205

Pro?Arg?Glu?Arg?Ala?Ser?Glu?Pro?Lys?His?Lys?Asn?Gln?Ala?Glu?Ile

210 215 220

Ala?Ala?Cys

225

Claims

1. the HLA-A2 restriction epi polypeptide in human phosphotidylethanolabinding binding protein 4 sources it is characterized in that described polypeptide has the inducing cytotoxic T cell killing activity, and the sequence of described polypeptide is TLFCQGLEV.

2. an isolating nucleic acid is characterized in that, the HLA-A2 restriction epi polypeptide in the described human phosphotidylethanolabinding binding protein of its coding claim 14 sources.

3. an antigen presenting cell is characterized in that, described antigen presenting cell is by the HLA-A2 restriction epi polypeptide sensitization in the described human phosphotidylethanolabinding binding protein of claim 14 sources.

4. antigen presenting cell as claimed in claim 3 is characterized in that, described antigen presenting cell is selected from down group: dendritic cell, scavenger cell, B cell, inoblast or endotheliocyte.

5. composition is characterized in that it contains:

(a) the HLA-A2 restriction epi polypeptide or the described antigen presenting cell of claim 3 in the described human phosphotidylethanolabinding binding protein of 0.001-99.99wt% claim 14 sources; With

(b) acceptable carrier, thinner or vehicle.

6. composition as claimed in claim 5 is characterized in that described composition is a pharmaceutical composition, and described carrier, thinner or vehicle are pharmaceutically acceptable carrier, thinner or vehicle.

7. the HLA-A2 restriction epi polypeptide in the described human phosphotidylethanolabinding binding protein of claim 14 sources or the purposes of the described antigen presenting cell of claim 3 is characterized in that, are used to prepare the medicine of prevention and treatment tumour.