CN1948342B

CN1948342B - HLA-A2 restriction epi polypeptide originated from post selection cancer gene hRabj and its application

Info

Publication number: CN1948342B
Application number: CN2005100305299A
Authority: CN
Inventors: 李楠; 孙伟红; 陈涛涌; 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: 2010-12-29
Anticipated expiration: 2025-10-14
Also published as: CN1948342A

Abstract

This invention provides a HLA-A2 restricted epi-position polypeptide come from candidate Oncogene hRabJ, and the use of this epi-position and associated recombination protein, coding nucleotide sequence, antigen presenting cell, combination matter, and specific Immune effector cell aiming directly at this epi-position is used to express hRabJ oncotherapy and prevention.

Description

HLA-A2 restriction epi polypeptide and application thereof that a kind of new candidate oncogene hRabJ originates

Technical field

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

Background technology

HRabJ (SEQ ID NO:12) is that (dendritic cell, DC) the cDNA library carries out finding a kind of novel full-length cdna in the process of large scale sequencing, login (sequence number is AF178983) in the GenBank/EMBL database from dendritic cell.HRabJ 273 the amino acid whose protein (SEQ ID NO:13) of encoding are the GTP hydrolysising proteases (GTPase) of a Rab sample.RabJ mainly is distributed in tissues such as testis, brain and ovary, can in conjunction with and the degraded GTP, in cell, may be distributed in the nuclear, (heatshock protein 70 HSP70) can form complex body with heat shock protein 70.

By analysis to the assignment of genes gene mapping, prlmary structure of protein, cell and the tissue distribution of RabJ, find that RabJ may represent a new small G-protein family, it is characterized in that in its protein is formed, comprising nuclear localization signal (the nuclearlocalization signal of three class functional domain: N ends (1-18 amino acid) and middle (210-216 amino acid), NLS), the J functional domain (217-273 amino acid) of intermediary Rab sample functional domain (19-209 amino acid) and C end, and higher homology is arranged with the Ras family molecule.Three class functional domains mediate the position of appraising and deciding of RabJ respectively; With extracellular signals-modulating kinases (extracellular signal-regulatedkinase, ERK1/2) kinases (ERK kinase, MEK1/2), protein kinase C (protein kinase C, PKC), phosphatidylinositol 3-kinase (phosphatidylinositol 3-kinase, P85 subunit PI3K), the interaction of P53 subunit; With functions such as HSC70 and Raf (Ras correlation factor, Ras-associated factor) interactions.The nucleotide sequence of RabJ and aminoacid sequence and method for making are disclosed among the Chinese patent application CN01126826.3.

In NIH3T3 clone, cross expressing of RabJ can promote cyclin to express, promote cell proliferation, promote the cell cycle progress, also can form fibrosarcoma in nude mouse, and prompting RabJ is the small G-protein of an oncogene sample.The expression of finding RabJ in tumour cell simultaneously is often very high, and the expression that suppresses RabJ can suppress the propagation of tumour cell and the oncogenic activity of inside and outside.Its mechanism of action may be the site by the karyon anchor as phosphorylation MEK1/2, suppresses the caryoplasm transposition of MEK1/2, thereby causes the continuous activation of MEK1/2 and promote the generation of tumour.

RT-PCR shows, hRabJ is the molecule more widely that distributes in tumour cell, at various human source hematopoietic cells such as HeLa, Jurkat, Molt-4, NB4, THP-1, U937, MCF-7, PC-3 is that higher expression is all arranged in leukemia cell and the solid tumor, does not but express in (as NIH3T3, TC-1) in normal clone.Blocking-up or inhibition hRabJ express and can suppress growth of tumor speed and reduce its tumorigenicity, thereby make hRabJ can be used as the target gene of oncotherapy or diagnosis.

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

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, be applied to antitumor and the 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 have two energy at least by HLA-A ^*0201 processes, presents, and becomes the new candidate of polypeptide vaccine research; (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, and positive rate accounts for the first place of each subgroup of MHC-I quasi-molecule between 40-60%, because HLA-A ^*The 0201st, the site that crowd's frequency of occurrences is the highest, 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 measure epi-position and HLA-A ^*One of instrument cell of 0201 molecule bonding force, it is the HLA-A of a strain antigen presentation transporter defective ^*0201 type cell strain, this cell surface is only expressed the HLA-A that does not contain the endogenous antigen molecule ^*0201 molecule, thus 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 hRabJ is provided the HLA-A2 restriction epi polypeptide in source, described polypeptide has the inducing cytotoxic T cell killing activity, and is selected from down group:

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

X _aa1-FVSKYLATI-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, R, KR or EKR.

And X _Aa2Be do not exist, G, GI or GID.

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 FVSKYLATI (SEQ ID NO:IDNO: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 hRabJ 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 hRabJ 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 hRabJ.

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 hRabJ 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 hRabJ source.

In a seventh aspect of the present invention, the purposes of the HLA-A2 restriction epi polypeptide in hRabJ 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 R41-49 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 R41-49 peptide sensitization.

Fig. 2: induce HLA-A2.1/K in dendritic cell (DC) body of R41-49 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 R41-49 peptide sensitization.

Embodiment

The inventor finds that through extensive and deep research the expression of RabJ in the tumour cell often raises, and the expression that suppresses RabJ can suppress the propagation of tumour cell and the oncogenic activity of inside and outside.In view of the above, the inventor is carrying out optionally having synthesized many energy and HLA-A on the basis of computer simulation analysis to hRabJ ^*0201 combination also induces body to produce the special antigen peptide of hRabJ of CTL, and pass through the T2 cell in conjunction with experiment, filters out and HLA-A ^*0201 has the epitope peptide of strong affinity, and its immunogenicity is estimated, and finds that it not only can be at HLA-A2.1/K ^bInduce specific, HLA-A in transgenic mice and the healthy human peripheral blood ^*0201 restrictive cytotoxic T cell, 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, RabJ mainly is distributed in testis tissue in primary and secondary spermatocyte (spermatocyte), the early stage spermatid (spermatids), in mesenchymal cell and sustenticular cell, do not have and express, similar with cyclin cyclin A and the expression pattern of cyclin D1 in mouse testis, prompting RabJ may be relevant with cell cycle regulating.

In NIH3T3 clone, cross expressing of RabJ can promote cyclin to express, promote cell proliferation, promote the cell cycle progress, and can promote this clone on soft agar, to form the clone, and in nude mouse, also can be formed into fibrosarcoma, pointing out it may be the small G-protein of an oncogene sample.

Research of the present invention finds that also the expression of RabJ often raises in tumour cell, and the expression that suppresses RabJ can suppress the propagation of tumour cell and the oncogenic activity of inside and outside.Because RabJ can form complex body with multiple protein, therefore suppress each kinase whose activity with kinase inhibitor, find that the MEK/ERK inhibitor can suppress the external tumorigenesis ability of RabJ fully, the signal transduction mechanism that prompting ERK1/2 relies on may be the tumorigenesis mechanism of RabJ.

The test-results of Laser Scanning Confocal Microscope is also pointed out, and RabJ is that the nuclear anchor of a MEK1/2 albumen, by increasing the level of nuclear phosphorylation MEK1/2, in the local continuous activation that forms ERK1/2 of karyon, causes the generation of tumour.

Therefore, the inventor studies show that, RabJ is as the small G-protein family member, in processes such as cell cycle regulating, cell proliferation, tumour generation, play an important role, its mechanism may be the effect of the MEK1/2 promotion ERK1/2 mediation of phosphorylation by the nuclear anchor, promotes the generation of cell proliferation and tumour.Therefore, RabJ is the small G-protein of an oncogene sample, can be used as potential candidate target in the diagnosis of clinical tumor and treatment.

In view of the above, the inventor is carrying out hRabJ on the basis of computer simulation analysis, has optionally synthesized many possible and HLA-A ^*0201 combination also induces body to produce the special antigen peptide of hRabJ of CTL,, filters out and HLA-A in conjunction with experiment by the T2 peptide ^*0201 has the epitope peptide of strong affinity, and its immunogenicity is estimated, and finds that it not only can be at HLA-A2.1/K ^bInduce specific, HLA-A in transgenic mice and the healthy human peripheral blood ^*0201 restrictive cytotoxic T cell, 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 candidate oncogene hRabJ 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 R41-49 (SEQ ID NO:1).In addition, also comprise having and the variant form R41-49 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 ` of this area, 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 R41-49.

A kind of polypeptide of preferably deriving is 1 or 2 or 3 or 4 amino acid (as R, KR, EKR or CEKR) of 37-40 position in the upstream of SEQ ID NO:1 is added corresponding to hRabJ, and/or in the downstream of SEQ ID NO:1 is added corresponding to

hRabJ

1 or 2 or 3 or 4 amino acid (as G, GI, GID or GIDY) of 50-53 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 RabJ 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

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

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

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 ⁵Individual cell/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, add the FITC mark the specific mAb BB7.2 of HLA-A2 (available from Sterotec Ltd, Oxford, UK), ice bath 45 minutes, PBS are washed the back and are detected average fluorescent strength with flow cytometer.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

Immunofluorescence technique detection of peptides and HLA-A ^*0201 molecule in conjunction with situation, 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, and expression amount that then can detected MHC-I quasi-molecule is many more, is to detect index with the average fluorescent strength.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 of the hRabJ dietary protein origin polypeptide that immunofluorescence technique records ^*0201 bonded avidity result is as shown in table 1.The inventor filters out the epi-position R41-49 (SEQ ID NO:1, sequence is TLFCQGLEV) with the HLA-A2 high-affinity from the polypeptide in candidate oncogene hRabJ source.

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

High-affinity: FI＞1

Embodiment 2 expresses the preparation of the adenovirus (pAdRabJ) of RabJ

With the plasmid that comprises people RabJ cDNA that obtained among Chinese patent 01126826.3 embodiment 1 is template, cut through the SalI-XhoI 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 people RabJ-pShuttle-CMV carrier of correct sequence after the PmeI 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.Confirm that through order-checking having recombinated has produced the adenovirus carrier that comprises people RabJ encoding sequence.

This adenovirus carrier utilizes Lipofactamine 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 (pAdRabJ) of RabJ, 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 hRabJ 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 R41-49 (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 ⁶Individual cell/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 ⁶Individual cell/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 respectively load epitope peptide R41-49, SSp-1 (irrelevant contrast, sequence is RLNEVAKNL (SEQ IDNO:11)) the T2 cell, express T2 (positive control) that the adenovirus (pAdRabJ) of RabJ infects and unloaded T2 cell as target cell, add ⁵¹Cr (buys from Amersham company 100 μ Ci/10 ⁶Individual cell), put in 37 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.Than the adding effector cell, 37 hatched 4 hours, collected each 100 μ l of each hole supernatant, detected the cpm value with the γ calculating instrument by 50: 1,25: 1,12.5: 1 three different targets of imitating.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 R41-49 of the HLA-A2 sensitization in hRabJ source can significantly be induced HLA-A2.1/K ^bTransgenic mice produces restricted polypeptid specificity cytotoxic T cell and killing activity, and can stimulate IFN-γ to produce.The test-results of R41-49 as depicted in figs. 1 and 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〉the HLA-A2 restriction epi polypeptide and the application thereof in a kind of new candidate oncogene hRabJ source

<130>057874

<160>13

<170>PatentIn?version?3.1

<210>1

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>1

Phe?Val?Ser?Lys?Tyr?Leu?Ala?Thr?Ile

1 5

<210>2

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Gly?Ile?Asp?Tyr?Gly?Val?Thr?Lys?Val

1 5

<210>3

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>3

Trp?Leu?Ala?Glu?Met?Lys?Gln?Glu?Leu

1 5

<210>4

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>4

Val?Ile?Ser?Met?Gly?Asn?Ala?Glu?Val

1 5

<210>5

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>5

Lys?Ala?Tyr?Arg?Lys?Leu?Ala?Val?Leu

1 5

<210>6

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>6

Val?Leu?Leu?His?Pro?Asp?Lys?Cys?Val

1 5

<210>7

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>7

Asn?Met?Glu?Asn?Ile?Ile?Phe?Val?Val

1 5

<210>8

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>8

Val?Val?Asn?Ala?Arg?Thr?Ala?Leu?Leu

1 5

<210>9

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>9

Asp?Met?Leu?Gly?Val?Lys?Pro?Gly?Ala

1 5

<210>10

<211>9

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>10

Tyr?Leu?Ser?Gly?Ala?Asn?Leu?Asn?Leu

1 5

<210>11

<211>9

<212>PRT

＜213〉contrast polypeptide

<400>11

Arg?Leu?Asn?Glu?Val?Ala?Lys?Asn?Leu

1 5

<210>12

<211>1787

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<220>

<221>CDS

<222>(25)..(843)

<223>

<400>12

caagcttatg?catgcggccg?ctga?atg?gag?gcc?aac?atg?ccg?aag?cgg?aag 51

Met?Glu?Ala?Asn?Met?Pro?Lys?Arg?Lys

1 5

gag?ccc?ggc?agg?tct?ctc?cgc?atc?aaa?gtc?arc?tcc?atg?ggc?aac?gcc 99

Glu?Pro?Gly?Arg?Ser?Leu?Arg?Ile?Lys?Val?Ile?Ser?Met?Gly?Asn?Ala

10 15 20 25

gaa?gtg?ggg?aaa?agc?tgt?att?ata?aag?cga?tac?tgt?gag?aaa?aga?ttc 147

Glu?Val?Gly?Lys?Ser?Cys?Ile?Ile?Lys?Arg?Tyr?Cys?Glu?Lys?Arg?Phe

30 35 40

gtg?tct?aaa?tac?ctg?gca?aca?att?gga?att?gac?tat?gga?gtc?aca?aag 195

Val?Ser?Lys?Tyr?Leu?Ala?Thr?Ile?Gly?Ile?Asp?Tyr?Gly?Val?Thr?Lys

45 50 55

gta?cac?gtc?aga?gac?aga?gaa?atc?aaa?gtt?aac?atc?ttt?gat?atg?gct 243

Val?His?Val?Arg?Asp?Arg?Glu?Ile?Lys?Val?Asn?Ile?Phe?Asp?Met?Ala

60 65 70

gga?cat?ccc?ttc?ttc?tat?gag?gtt?cga?aat?gag?ttt?tac?aag?gac?aca 291

Gly?His?Pro?Phe?Phe?Tyr?Glu?Val?Arg?Asn?Glu?Phe?Tyr?Lys?Asp?Thr

75 80 85

cag?ggt?gtg?ata?ctg?gtc?tat?gat?gtt?ggg?cag?aaa?gac?tcc?ttt?gac 339

Gln?Gly?Val?Ile?Leu?Val?Tyr?Asp?Val?Gly?Gln?Lys?Asp?Ser?Phe?Asp

90 95 100 105

gcc?ctt?gat?gcg?tgg?ctg?gca?gaa?atg?aag?caa?gag?ctt?gga?cct?cat 387

Ala?Leu?Asp?Ala?Trp?Leu?Ala?Glu?Met?Lys?Gln?Glu?Leu?Gly?Pro?His

110 115 120

gga?aac?atg?gaa?aat?att?ata?ttt?gta?gtt?tgt?gcc?aac?aag?att?gat 435

Gly?Asn?Met?Glu?Asn?Ile?Ile?Phe?Val?Val?Cys?Ala?Asn?Lys?Ile?Asp

125 130 135

tgt?acc?aaa?cat?cgc?tgt?gta?gat?gaa?agt?gaa?gga?cgt?ctt?tgg?gct 483

Cys?Thr?Lys?His?Arg?Cys?Val?Asp?Glu?Ser?Glu?Gly?Arg?Leu?Trp?Ala

140 145 150

gaa?agc?aaa?ggg?ttc?ctg?tac?ttt?gaa?act?tca?gca?caa?act?gga?gaa 531

Glu?Ser?Lys?Gly?Phe?Leu?Tyr?Phe?Glu?Thr?Ser?Ala?Gln?Thr?Gly?Glu

155 160 165

ggc?att?aat?gag?atg?ttc?cag?acc?ttt?tat?ata?tcc?ata?gtt?gat?tta 579

Gly?Ile?Asn?Glu?Met?Phe?Gln?Thr?Phe?Tyr?Ile?Ser?ILe?Val?Asp?Leu

170 175 180 185

tgt?gaa?aat?ggc?ggg?aaa?cgc?cct?acc?acc?aat?agc?agt?gct?agt?ttc 627

Cys?Glu?Asn?Gly?Gly?Lys?Arg?Pro?Thr?Thr?Asn?Ser?Ser?Ala?Ser?Phe

190 195 200

acc?aaa?gaa?caa?gca?gat?gcc?att?cgc?aga?att?cga?aat?agt?aaa?gac 675

Thr?Lys?Glu?Gln?Ala?Asp?Ala?Ile?Arg?Arg?Ile?Arg?Asn?Ser?Lys?Asp

205 210 215

agt?tgg?gac?atg?ctg?gga?gtc?aaa?cct?ggg?gcc?tca?agg?gat?gaa?gtc 723

Ser?Trp?Asp?Met?Leu?Gly?Val?Lys?Pro?Gly?Ala?Ser?Arg?Asp?Glu?Val

220 225 230

aat?aaa?gcg?tat?cgg?aaa?ctt?gct?gtg?ctt?ctt?cac?cct?gac?aaa?tgt 771

Asn?Lys?Ala?Tyr?Arg?Lys?Leu?Ala?Val?Leu?Leu?His?Pro?Asp?Lys?Cys

235 240 245

gta?gca?cct?ggc?agt?gaa?gat?gcc?ttc?aaa?gca?gtt?gtg?aat?gct?cgg 819

Val?Ala?Pro?Gly?Ser?Glu?Asp?Ala?Phe?Lys?Ala?yal?Val?Asn?Ala?Arg

250 255 260 265

aca?gcc?ctc?ctg?aaa?aac?atc?aag?tagaaagtac?agaaaaaagc?cacatgtggg 873

Thr?Ala?Leu?Leu?Lys?Asn?Ile?Lys

270

actcaaatgc?aaacagactt?tccctagagg?tgaaataacc?aacgtggagt?tttccttccc 933

agaatctcac?tgctcttttc?attcatgtgt?tgtcatttgt?atatcagtaa?ttcaggtacc 993

catttcatag?acattttact?gagaaatgac?ctgcatttgt?atgaagtgaa?ctgagcgtca 1053

caccctgtac?ttcatttcat?atttctagat?aattctgaat?ttttttctca?ttcgtcagct 1113

ctgtaattat?agtatcactt?agacatttca?cttggggaaa?tccacaaggt?tcctggaggg 1173

agggaagaga?ggacaagagg?accctttcac?tttttctttt?ttacggaatt?catcatcaga 1233

gaagaaaata?acaaaaatgg?aagcaaacaa?catcagaacc?cctgtaagtt?tggtgtgacc 1293

ttacagacaa?gttgctgctt?ttacaatgag?ttccttaggt?ggtattttaa?cccatcgatc 1353

tataatgatg?actcttggca?gccctttggg?agtttgtaaa?atgaggtgat?acagttctga 1413

attgagcatt?cctttatgat?attcactctg?ttcctcttct?gcagccacca?gtgggagaga 1473

caagccagtc?ctaagagaaa?aggtggtggc?agccacaaat?tctaggtaca?ctggctgctg 1533

cctatcctgt?ccctggatct?gaggcctttc?ccttgccata?gaaatggttg?ctggtagcag 1593

tagagagcac?tgtgcacctg?ggaatgagga?atcaggcccc?aagacagaag?tacttggagg 1653

agccagctgc?agtagtatcc?gcctgtagtc?ccagctactc?aggaggctga?gacaggagga 1713

ttgcttaagc?ccaggagctc?aagtcccacc?tgggcaacat?agtaagatct?tgtctcttaa 1773

aaaaaaaaaa?aaaa 1787

<210>13

<211>273

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>13

Met?Glu?Ala?Asn?Met?Pro?Lys?Arg?Lys?Glu?Pro?Gly?Arg?Ser?Leu?Arg

1 5 10 15

Ile?Lys?Val?Ile?Ser?Met?Gly?Asn?Ala?Glu?Val?Gly?Lys?Ser?Cys?Ile

20 25 30

Ile?Lys?Arg?Tyr?Cys?Glu?Lys?Arg?Phe?Val?Ser?Lys?Tyr?Leu?Ala?Thr

35 40 45

Ile?Gly?Ile?Asp?Tyr?Gly?Val?Thr?Lys?Val?His?Val?Arg?Asp?Arg?Glu

50 55 60

Ile?Lys?Val?Asn?Ile?Phe?Asp?Met?Ala?Gly?His?Pro?Phe?Phe?Tyr?Glu

65 70 75 80

Val?Arg?Asn?Glu?Phe?Tyr?Lys?Asp?Thr?Gln?Gly?Val?Ile?Leu?Val?Tyr

85 90 95

Asp?Val?Gly?Gln?Lys?Asp?Ser?Phe?Asp?Ala?Leu?Asp?Ala?Trp?Leu?Ala

100 105 110

Glu?Met?Lys?Gln?Glu?Leu?Gly?Pro?His?Gly?Asn?Met?Glu?Asn?Ile?Ile

115 120 125

Phe?Val?Val?Cys?Ala?Asn?Lys?Ile?Asp?Cys?Thr?Lys?His?Arg?Cys?Val

130 135 140

Asp?Glu?Ser?Glu?Gly?Arg?Leu?Trp?Ala?Glu?Ser?Lys?Gly?Phe?Leu?Tyr

145 150 155 160

Phe?Glu?Thr?Ser?Ala?Gln?Thr?Gly?Glu?Gly?Ile?Asn?Glu?Met?Phe?Gln

165 170 175

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

180 185 190

Pro?Thr?Thr?Asn?Ser?Ser?Ala?Ser?Phe?Thr?Lys?Glu?Gln?Ala?Asp?Ala

195 200 205

Ile?Arg?Arg?Ile?Arg?Asn?Ser?Lys?Asp?Ser?Trp?Asp?Met?Leu?Gly?Val

210 215 220

Lys?Pro?Gly?Ala?Ser?Arg?Asp?Glu?Val?Asn?Lys?Ala?Tyr?Arg?Lys?Leu

225 230 235 240

Ala?Val?Leu?Leu?His?Pro?Asp?Lys?Cys?Val?Ala?Pro?Gly?Ser?Glu?Asp

245 250 255

Ala?Phe?Lys?Ala?Val?Val?Asn?Ala?Arg?Thr?Ala?Leu?Leu?Lys?Asn?Ile

260 265 270

Lys

Claims

1. the HLA-A2 restriction epi polypeptide in a hRabJ source is characterized in that described polypeptide has the inducing cytotoxic T cell killing activity, and for comprising the polypeptide of the aminoacid sequence in the following general formula I:

X _aa1-FVSKYLATI-X _aa2 (I)

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

2. polypeptide as claimed in claim 1 is characterized in that, described polypeptide is FVSKYLATI.

3. a recombinant protein is characterized in that, it contains the HLA-A2 restriction epi polypeptide in claim 1 or 2 described hRabJ sources.

4. an isolating nucleic acid is characterized in that, the HLA-A2 restriction epi polypeptide in its coding claim 1 or 2 described hRabJ sources.

5. an antigen presenting cell is characterized in that, described antigen presenting cell is by the HLA-A2 restriction epi polypeptide sensitization in claim 1 or 2 described hRabJ source.

6. antigen presenting cell as claimed in claim 5 is characterized in that, described antigen presenting cell is selected from down group: dendritic cell, scavenger cell, B cell, inoblast, endotheliocyte.

7. composition is characterized in that it contains:

(a) HLA-A2 restriction epi polypeptide or the claim 5 or the 6 described antigen presenting cells in 0.001-99.99wt% claim 1 or 2 described hRabJ sources; With

(b) acceptable carrier, thinner or vehicle.

8. composition as claimed in claim 7 is characterized in that described composition is a pharmaceutical composition, and described carrier, thinner or vehicle are pharmaceutically acceptable carrier, thinner or vehicle.

9. a claim 1 or the HLA-A2 restriction epi polypeptide in 2 described hRabJ sources or the purposes of claim 5 or 6 described antigen presenting cells is characterized in that, are used to prepare the medicine of prevention and treatment tumour.