CN1223689A - Brain glycogen phosphorylase cancer antigen - Google Patents

Abstract

The invention describes brain glycogen phosphorylase tumor rejection antigen precursors, including nucleic acids encoding such tumor rejection antigen precursors, tumor rejection antigen peptides or precursors thereof and antibodies relating thereto. Methods and products also are provided for diagnosing and treating conditions characterized by expression of a brain glycogen phosphorylase tumor rejection antigen or precursor thereof.

Description

Brain glycogen phosphorylase cancer antigen

Technical field of the present invention

The present invention relates to Tumor rejection antigen and its precursor.Relate in particular at least a Tumor rejection antigen that Tumor rejection antigen is provided by the HLA molecule.The invention still further relates to the nucleic acid molecule of codes for tumor rejection antigen and its precursor.Particularly, these nucleic acid molecule and by its encoded protein matter and peptide derivant thereof, relevant antibody and cytotoxic lymphocyte in the application aspect diagnosis and the treatment.

Prior art of the present invention

Mammals is very complicated to the immunity system of discerning and reacting of foreign matter and xenobiotic.An important aspect of this system is a t cell responses.The T cell can discern other cell and with they reactions, this be by to the complex body of the peptide of those cell surfaces and molecule, promptly be called human leucocyte antigen (HLA) (" HLA ") or major histocompatibility complex (" MHCs ") is realized.These peptides are to derive out from bigger molecule, and the cell that has the HLA/MHC molecule also has these bigger molecules.Referring to people such as Male at Advanced Immunology (J.P.Lipincott Company, 1987) book, particularly 6-10 chapter.The interaction of T cell and HLA/ peptide complex body is restrictive, and promptly specific T cell is specific to the complex body of specific HLA molecule and peptide.If do not have specific T cell to exist,, t cell responses can not take place yet although the existence of its corresponding complex body is then arranged.Similarly, if there is not the existence of specific complex body,, do not have t cell responses although the existence of T cell is then arranged yet.This mechanism is present in the reaction of immunity system to allogenic material, is present in the autoimmunity pathology, and is present among the unusual reaction of pair cell.

The mechanism of T cell recognition allogenic material also is present in the cancer.Described have some directly opposing clone from the molten born of the same parents' property T of the cell of autologous melanoma lymphocyte (CTL).In the PCT application PCT/US92/04354 that submitted on November 26th, 1992, disclosed the gene of gang's tumour-specific, i.e. " MAGE " family.These expression of gene products are integrated in the peptide, and then, these peptides are expressed by cell surface again.。Can cause the molten born of the same parents to tumour cell like this by CTLs.It is said these genes " tumor rejection antigen precursor " i.e. " TRAP " molecule of can encoding, and the peptide that they derive out is called as " Tumor rejection antigen " i.e. " TRAs ".The details of the gene of family hereto can be referring to people such as Traversari in Immunogenetics35:145 (1992), and people such as van der Bruggen are at the document of Science 254:1643 (1991).Can also be referring to United States Patent (USP) the 5th, 342, No. 774.

At United States Patent (USP) the 5th, 405, instructed the MAGE nonapeptide in No. 940, they are carried by the HLA-A1 molecule.Owing to know concrete peptide corresponding to concrete this specific specificity of HLA molecule, so what should predict is that certain peptide only combines with a kind of HLA molecule, and does not combine with other HLA molecule.This point is extremely important, because different individualities has different HLA phenotypes.The result is, though identify a certain peptide be a certain concrete HLA molecule to as if having diagnosis and a treatment result,, they are only to having the individual effective of the sort of specific HLA phenotype.Because cellular abnormality is not limited to a certain concrete HLA phenotype, and needed diagnosis and treatment all need the phenotype of this cellular abnormality is known about, so, need in this field, carry out more deep work.

Be found that in addition the MAGE expression product is integrated among second kind of TRA.It is entrained that this 2nd TRA is cloned 10 molecules by HLA-C.Therefore, a certain TRAP can the multiple TRAs of output.

In disclosed PCT application on July 7th, 1994 WO94/14459, tyrosine oxidase is described to tumor rejection antigen precursor.This piece document illustration the molecule that produces by some normal cells (as melanophore) be integrated in the tumour cell, produce by the entrained Tumor rejection antigen of HLA-A2 molecule.

In disclosed PCT application on September 29th, 1994 WO94/21126, having disclosed is not the 2nd TRA from tyrosinase derived, and it is carried by the HLA-A2 molecule.This TRA is from the TRAP deutero-, still, and its non-MAGE gene of encoding, and be called melanocyte-A (Melan-A).This piece document points out that a certain concrete HLA molecule can provide different sources deutero-TRA.

In disclosed PCT application on January 5 nineteen ninety-five WO95/00159, disclosed another kind of incoherent tumor rejection antibody precursor, be called " BAGE " precursor.Also described from TRAP deutero-TRA simultaneously.They and MHC molecule HLA-C-clone 10 form complex body.

In disclosed PCT application on February 2 nineteen ninety-five WO95/03422, disclosed the tumor rejection antigen precursor that another kind is called " GAGE ".GAGE precursor and BAGE or MAGE are incoherent.

Work described in above-mentioned each piece document, patent or the patent application, major part are all about MAGE gene, BAGE gene and each family of GAGE gene.These genes are expressed in some tumours, but all are dormancy in other the normal cell except testis.And they are not expressed in kidney.

Recently, found the tumor rejection antigen precursor of a kind of incoherent being called " RAGE " again.Their characteristics are to express in kidney.These RAGE precursors are not relevant with GAGE, BAGE or each family of MAGE.

The brain glycogen phosphorylase gene is expressed in normal grownup's brain and retinal pigment epithelium.Report in the past is that these genes have overexpression in some kidneys, liver cancer and cancer of the stomach.But, report that in addition the brain glycogen phosphorylase gene can excite to have has specific breeding from body CTL to brain glycogen phosphorylase peptide and HLA complex body.In other words, brain glycogen phosphorylase is not known TRAP.

What find now is that the brain glycogen phosphorylase gene is to express in the tumour cell of melanoma cells and some other type.But also discovery is brain glycogen phosphorylase genes encoding Tumor rejection antigen and its precursor.The brain glycogen phosphorylase gene does not show the homology with MAGE gene cluster, BAGE gene cluster, GAGE gene cluster or RAGE gene cluster.

Specific descriptions of the present invention are as follows.

General introduction of the present invention

The invention provides the brain glycogen phosphorylase fragment that is separated.The present invention also provides separated nucleic acid molecule, has contained the expression vector of these molecules, and by the host cell of these molecule transfections.Above-mentioned these materials and brain glycogen phosphorylase itself may be used to express in the diagnosis and treatment of disease that brain glycogen phosphorylase TRA or TRAP are feature.

According to an aspect of the present invention, provide the brain glycogen phosphorylase of separating.The aminoacid sequence that it comprises No. 15 aminoacid sequence (SEQ ID NO:15) and is no more than 75% No. 21 (SEQ ID NO:21).In some embodiments, these fragments of separating comprise no more than 100 amino acid.In other embodiments, these fragments that are separated can comprise the molecule with No. 15, No. 14, No. 13, No. 12 or No. 5 aminoacid sequence.In the other embodiment, this fragment that is separated comprises the molecule with No. 14, No. 13 or No. 12 aminoacid sequence.

According to a further aspect in the invention, provide the nucleic acid molecule that is separated.This nucleic acid molecule encoding is selected from the peptide in a group that the various fragment of above-mentioned brain glycogen phosphorylase forms.Preferably, the molecule that contains of the coded peptide of these nucleic acid molecule has comprised No. 15, No. 14, No. 13, No. 12 or No. 5 aminoacid sequence.In other embodiments, these peptides have the aminoacid sequence that is selected from by in a group that is formed for No. 14, No. 13 and No. 12.

According to another aspect of the present invention, expression vector and the host cell that contains these carriers are also provided.These expression vectors can contain one or more the above-mentioned nucleic acid molecule that is separated.In a specific embodiment, expression vector comprises the nucleic acid of No. 14, No. 13 or No. 12.Other expression vector according to the present invention comprises that above-mentioned nucleic acid that is separated and encoding those can offer brain glycogen phosphorylase Tumor rejection antigen of the present invention the nucleic acid of the HLA molecule of molten born of the same parents T cell.Their example is HLA-A2.And described host cell can be expressed the HLA molecule in endogenous ground, for example expresses HLA-A2.

According to another aspect of the present invention, provide optionally T cell to strengthen its method to molten born of the same parents' property T cell-specific of brain glycogen phosphorylase Tumor rejection antigen to a certain colony.This method comprises, allows the T cell of a certain colony contact with the preparation that provides the brain glycogen phosphorylase Tumor rejection antigen with the complex body of the molecule that HLA is provided.Must be enough to optionally strengthen the molten born of the same parents T cell of the T cell colony that is separated with the consumption of the preparation of T cells contacting.In certain embodiments, the molecule that HLA is provided is HLA-A2, and the brain glycogen phosphorylase Tumor rejection antigen is the peptide that comprises No. 15 aminoacid sequence.In the other embodiment, peptide is No. 21 7-100 successive amino acid in the aminoacid sequence.In preferred embodiments, the molecule of this peptide has the sequence that is selected among a group that is made up of No. 15, No. 14, No. 13, No. 12 and No. 5 aminoacid sequence.More preferably, the sequence that these peptides contained comprises No. 14, No. 13 or No. 12 aminoacid sequence.

The brain glycogen phosphorylase peptide is provided by the HLA molecule and helps medical diagnosis on disease by this understanding of CTLs identification.Therefore, according to another aspect of the present invention, provide being the diagnostic method of the disease of feature to express the brain glycogen phosphorylase Tumor rejection antigen.This method comprises the biological sample that will separate from the patient on one's body and is that specific preparation contacts to the brain glycogen phosphorylase Tumor rejection antigen.This biological sample is separated from the tissue of non-brain, non-retinal pigment epithelium.Then, this method diagnoses the illness by determining the interaction between said preparation and the brain glycogen phosphorylase Tumor rejection antigen.In one embodiment, the brain glycogen phosphorylase Tumor rejection antigen is a kind of peptide that comprises No. 15 aminoacid sequence.In another embodiment, this peptide comprises in No. 21 aminoacid sequence the amino acid between 7-100 successive amino acid and No. 15 sequence.Preferably, the sequence that contains of this peptide molecule is selected among one group that is made up of No. 15, No. 14, No. 13, No. 12 and No. 5 sequence.Most preferably, this sequence is No. 14, No. 13 or No. 12 aminoacid sequence.

Above-mentioned method provides there being the methods for the diagnosis of diseases of brain glycogen phosphorylase TRAs for the basis.It is the methods for the diagnosis of diseases of feature that another aspect of the present invention provides to express brain glycogen phosphorylase Tumor rejection antigen (itself and HLA molecule form complex body).In some embodiments, complex body wherein forms with HLA-A2.This method comprises the biological sample that will separate from the patient on one's body with contacting with complex body bonded preparation, and disease is determined in the interaction between definite then complex body and the preparation.In one embodiment, the brain glycogen phosphorylase Tumor rejection antigen is the peptide that comprises No. 15 aminoacid sequence.Preferably, the sequence that contains of this peptide molecule is selected among one group that is made up of No. 15, No. 14, No. 13, No. 12 and No. 5 sequence.In other preferred embodiment, the sequence that the molecule that this peptide comprises has is to be selected among one group that is made up of No. 14, No. 13 or No. 12 aminoacid sequence.

According to another aspect of the present invention, provide with express brain glycogen phosphorylase or its coding nucleic acid be feature methods for the diagnosis of diseases.This method comprises the adenocarcinoma tissue of the non-brain from the patient, non-retinal pigment epithelium, non-renal cell carcinoma, non-liver cancer and non-cancer of the stomach isolates biological sample.In some embodiments, this method comprises this biological sample and contacts with brain glycogen phosphorylase bonded preparation, and diagnoses the illness by the interaction between definite brain glycogen phosphorylase and the said preparation.In other embodiments, this method comprises that be that specific preparation contacts with biological sample with nucleic acid to coding brain glycogen phosphorylase or its expression product.Interactional mensuration between preparation and nucleic acid or its expression product is used to diagnose the illness.

Except diagnosing the illness, also need some diseases is treated.According to another aspect of the present invention, provide being the disease patient's of feature methods of treatment to express the brain glycogen phosphorylase Tumor rejection antigen.This method comprises the preparation that doses is provided to the patient, and this dosage can optionally strengthen the complex body of patient's HLA and brain glycogen phosphorylase Tumor rejection antigen and reach the level of improving disease.Preferably, this complex body is formed by HLA-A2 and brain glycogen phosphorylase Tumor rejection antigen.Preferably, peptide is to contain the molecule that is selected from the sequence among a group that is made up of No. 15, No. 14, No. 13, No. 12 and No. 5 sequence to be formed.In other embodiment preferred, this peptide is formed by having the molecule that is selected from the aminoacid sequence in a group that is formed with No. 14, No. 13 or No. 12 aminoacid sequence.Another kind method comprise the patient who receives treatment to needs provide the dosage that is enough to improve disease from the molten born of the same parents T of body cell, wherein be to be specific from the molten born of the same parents T of body cell to HLA molecule and the formed complex body of brain glycogen phosphorylase Tumor rejection antigen.Preferably, this complex body is formed by HLA-A2 and aforesaid brain glycogen phosphorylase peptide.

According to another aspect of the present invention, provide the nucleic acid that comprises separated coding brain glycogen phosphorylase Tumor rejection antigen or contain the application of preparation aspect the preparation medicine of the polypeptide of brain glycogen phosphorylase Tumor rejection antigen.Said preparation optionally strengthen the patient by HLA and the formed complex body of brain glycogen phosphorylase Tumor rejection antigen.In some embodiments, the brain glycogen phosphorylase Tumor rejection antigen is the peptide that comprises No. 15 aminoacid sequence.Preferably, the sequence that contains of this peptide molecule is selected among one group that is formed with No. 15, No. 14, No. 13, No. 12 and No. 5 sequence.More preferably, the molecule that comprises of this sequence has and is selected among one group that is formed with No. 14, No. 13 and No. 12 aminoacid sequence.In addition, said preparation can comprise or not comprise that HLA carries molecule, the formed complex body of nucleic acid of this molecule and brain glycogen phosphorylase Tumor rejection antigen or this molecule of encoding.

According to another aspect of the present invention, provide complex body to HLA molecule and brain glycogen phosphorylase Tumor rejection antigen be specific from the molten born of the same parents T of body cell in the application aspect the preparation medicine.Preferably, it is HLA-A2 that this HLA carries molecule, and the brain glycogen phosphorylase Tumor rejection antigen is the peptide that comprises the molecule with No. 15 aminoacid sequence.In certain embodiments, this peptide is the successive amino acid of the 7-100 of No. 21 aminoacid sequence.Preferably, the sequence that contains of the molecule that comprises of this peptide is selected among one group that is formed with No. 15, No. 14, No. 13, No. 12 and No. 5 sequence.More preferably, the molecule that comprises of this peptide has the aminoacid sequence that is selected from a group that is formed with No. 14, No. 13 and No. 12 aminoacid sequence.

Another aspect of the present invention provides the pharmaceutical composition that contains above-mentioned preparation and/or cell.In one embodiment, said composition comprises brain glycogen phosphorylase or its fragment in conjunction with the HLA molecule of pharmacology effective dose, and pharmacology acceptable diluent, carrier or vehicle.In some embodiments, the HLA molecule is HLA-A2.Preferably, brain glycogen phosphorylase or its fragment comprise the amino acid whose peptide with No. 15 aminoacid sequence.In other embodiments, this pharmaceutical composition comprise the complex body to HLA molecule and brain glycogen phosphorylase Tumor rejection antigen of pharmacology effective dose be specific be separated from the molten born of the same parents T of body cell.

According to another aspect of the present invention, provide brain glycogen phosphorylase or the application of its fragment in medication preparation that is separated.These fragments comprise aminoacid sequence No. 15.The fragment of preferred brain glycogen phosphorylase molecule as mentioned above.In some embodiments, these medicines are oral medicine, suction medicine and injectable drug.

According to a further aspect in the invention, provide the application in the medicine of preparation treatment cancer of brain glycogen phosphorylase or its fragment.

According to a further aspect in the invention, provide kit.In kit, include aforesaid two kinds of materials at least and be separated to place.The composition that can also add if desired, other.In some embodiments, comprise the nucleic acid molecule that is separated of coding brain glycogen phosphorylase TRAP in this kit or contain the molecule of brain glycogen phosphorylase TRA, and form complex body and stimulate the HLA of molten born of the same parents' property t cell responses to carry molecule with TRA.A kind of coding that comprises in such kit contains the nucleic acid molecule of the peptide of No. 12, No. 13 or No. 14 sequence, and the nucleic acid molecule of coding HLA-A2.Another kind of kit according to the present invention is a kind of expression kit, it comprises the nucleic acid molecule that is separated of coding brain glycogen phosphorylase TRAP or TRA, or comprise the expression vector of brain glycogen phosphorylase TRAP or TRA coding nucleic acid molecule, and the nucleic acid molecule of coding HLA-A2.In some embodiments, this kit comprises that expressing those carries the host cell of the HLA molecule of brain glycogen phosphorylase TRAP or TRA.

Relevant with the nucleic acid molecule of above-mentioned any coding brain glycogen phosphorylase Tumor rejection antigen that is separated is, the present invention also comprises their degraded product, and the difference between their codon sequence and the nucleic acid that is separated only be genetic codon by degeneration, perhaps they are complementary sequences of above-mentioned any nucleic acid molecule.

The present invention also comprises the functional variant and the Equivalent of above-mentioned various molecules.

The detailed description that these purposes of the present invention and other purpose are all incited somebody to action below partly gives further instruction.

Brief description of drawings

Fig. 1 has represented the molten born of the same parents of CTL to the T2 cell, and assists with brain glycogen phosphorylase deutero-peptide.

Fig. 2 has represented the molten born of the same parents of CTL to the T2 cell, and assists with brain glycogen phosphorylase deutero-peptide.

Fig. 3 is the expression of brain glycogen phosphorylase in healthy tissues and tumor tissues.

Fig. 4 has represented the molten born of the same parents of CTL to the T2 cell, and uses from the brain glycogen phosphorylase deutero-peptide of brain, muscle and liver and assist.

Fig. 5 is the molten born of the same parents experiment of CTL to various clones, assists with the peptide of No. 14 sequence.

Concise and to the point description to sequence

No. 1 sequence:, can confirm the antigenic expression of being discerned by CTL246/7 with the shortest brain glycogen phosphorylase fragment of exonuclease preparation.

No. 2 sequence: with the brain glycogen phosphorylase fragment of 161 positions end.

No. 3 sequence: with the brain glycogen phosphorylase fragment of 140 positions end.

No. 4 sequence: 17mer peptide (aa18-33 of brain glycogen phosphorylase).

No. 5 sequence: 11mer peptide (aa1-11) is derived from No. 4 sequence.

No. 6 sequence: 10mer peptide (aa3-12) is derived from No. 4 sequence.

No. 7 sequence: 10mer peptide (aa4-13) is derived from No. 4 sequence.

No. 8 sequence: 10mer peptide (aa5-14) is derived from No. 4 sequence.

No. 9 sequence: 10mer peptide (aa6-15) is derived from No. 4 sequence.

No. 10 sequence: 10mer peptide (aa7-16) is derived from No. 4 sequence.

The o.11 sequence: 10mer peptide (aa8-17) is derived from No. 4 sequence.

No. 12 sequence: 10mer peptide (aa2-11) is derived from No. 5 sequence.

No. 13 sequence: 9mer peptide (aa3-11) is derived from No. 5 sequence.

No. 14 sequence: 8mer peptide (aa4-11) is derived from No. 5 sequence.

No. 15 sequence: 7mer peptide (aa5-11) is derived from No. 5 sequence.

No. 16 sequence: 6mer peptide (aa6-11) is derived from No. 5 sequence.

No. 17 sequence: the meaningful primer that the specific PCR of brain glycogen phosphorylase is amplified.

No. 18 sequence: the meaningless primer that the specific PCR of brain glycogen phosphorylase is amplified.

No. 19 sequence: the 11mer peptide of liver glycogen phosphorylase.

No. 20 sequence: the 11mer peptide of muscle glycogen phosphorylase.

No. 21 sequence: the full length sequence of brain glycogen phosphorylase cDNA.

No. 22 sequence: the translation product of No. 21 sequence.

No. 23 sequence: the nucleic acid of No. 14 sequence of encoding.

No. 24 sequence: the nucleic acid of No. 13 sequence of encoding.

No. 25 sequence: the nucleic acid of No. 12 sequence of encoding.

Of the present invention being described in detail

Can by antibody recognition on melanoma, by the HLA-A2 enzyme cut from body CTL, by the coded by said gene of just knowing in the past, i.e. brain glycogen phosphorylase.In the various healthy tissuess that were verified, this gene all is tranquillization in pcr analysis, and except brain and retinal pigment epithelium, and it is expressed in some tumor samples.

Embodiment 1

To description from the melanoma ctl clone of patient LB373

Tumour is that LB373-MEL is the melanoma cell series of deriving out from the tumor sample that is called LB373 patient.This sample is carried out radiation does not breed it.Then these are used for it is had the separation of specific molten born of the same parents T cell clone (" CTLs ") by the radiating cell.

Take peripheral blood lymphocyte (" PBLs ") sample on one's body from patient LB373, contact then with by the radiating melanoma cells.After 14 days, observe the molten born of the same parents to melanoma cells wherein, this shows that existing the complex body that the entrained peptide of tumour cell and HLA are formed in this sample is specific CTLs.

Molten born of the same parents' check of being adopted is to discharge check according to people such as Herin at the chromium described in the Int.J.Cancer 39:390-396 (1987).To being briefly described as follows of this check.The target melanoma cells is suspended in 10 then in growth in vitro ⁷In Dulbecco ' the s Modified Eagles substratum of individual cells/ml (DMEM), replenish in the substratum, cultivated 45 minutes at 37 ℃ with 30%FCS, and with the Na of 200 μ Ci/ml ( ⁵¹Cr) O ₄Mark.The cell that is labeled is given a baby a bath on the third day after its birth inferior with DMEM.Be suspended in then among the DMEM, replenish serum and 10% calf serum (FCS), after this, 10 microlitres are contained 10 with 10mM people ³The sample aliquot of individual cell joins in the droplet dish in 96 holes.Lymphocytic sample is placed in the same medium of 100 microlitres and joins each hole, and this check is carried out with double.To this droplet dish under 100g centrifugal 4 minutes, cultivate under the air of 8% carbonic acid gas, 37 ℃, 4 hours.

Centrifugal to the droplet dish once more, collect the supernatant and the counting of the sample aliquot of 100 microlitres.Discharge ⁵¹The percentage of Cr is calculated as follows:

ER wherein is in the observed experiment ⁵¹The release of Cr, SR be carry out simultaneously in 200 microlitre substratum, cultivate 10 ³The release of the cell of mark is measured, and MR is maximum release, obtains by add the 0.3%Triton X-100 of 100 microlitres in the target cell.

It is extended that those have showed the active monokaryon blood sample of high CTL, clones by limited dilution, and make the screening once more that uses the same method.Therefore, obtained first ctl clone.After being cloned in, this is called 246/76.

When stimulating with autologous tumor cell, ctl clone 246/76 produces TNF.Those performances have the melanoma cell series of the class at least of melanoma cell series LB373-MEL and discern test with ctl clone 246/76.After 4 hours, the clone of those shared HLA-A2 molecules is discerned by CTL.Conclusion is that CTL246/76 identification is by the entrained antigen of HLA-A2.

Embodiment 2

The cDNA clone's of the antigenic expression that guidance is discerned by CTL246/76 separation A, cDNA storehouse

Isolation of RNA from LB373-MEL gathers-A+RNA in conjunction with purifying with widow-dT.With the widow of containing Not I site-dT primer carry out reverse transcription, then synthetic second chain prepare cDNA (Superscrip Choice System, BRL, LifeTechnologies).Then cDNA is connected with Bst XI connector, cut with Not I enzyme, according to size packets (Sephacryl S-500 HR columns.BRL.LifeTechnologies), be cloned into Bst XI and the Not I site (Invitrogen) of pcDNA-I-Amp non-directional.The plasmid electroporation that to recombinate then is in TOP10F ' intestinal bacteria.700 ponds of 100 recombinant bacterias are exaggerated, and the plasmid DNA in each pond is extracted with the molten born of the same parents of alkali, and the potassium acetate precipitation is used phenol extraction.The transfection of B, pair cell and to the evaluation of cDNA

The COS cell of most CTL identification is only by the HLA-A2 transfection.Therefore, the cell of preparing other provides the peptide of being discerned by CTL246/76.

In order to separate the cDNA of the peptide that coding discerned by CTL246/76, two cell systems have been used.The HeLa cell (after this being called the HOB cell) of expressing the BK virus large T antigen can provide by the peptide of contrast cDNA coding, its level similar in the COS cell observed level: the tyrosine oxidase cDNA that dilutes in 200 uncorrelated cDNAs can be discerned by the CTLs of antityrosinase.Second kind of cell system available from Invitrogen company (San Diego, CA).The 293-EBNA-1 cell can be provided in the tyrosine oxidase cDNA deutero-peptide of cloning among the pCEP4, even also is like this under with the situation of uncorrelated cDNAs with dilution in 1: 800.This second cell system is used to the cDNA that the definite HOB of use cell system is separated.

Double is carried out in transfection to the HOB cell.In brief, with the inoculation of HOB cell sample, be inoculated in the flat tissue culture droplet dish with 15,000 cells/well, substratum is the DMEM that has replenished 10% calf serum.37 ℃ of following pair cell overnight incubation, remove substratum, change DMEM substratum with 100 microlitres/hole cumulative volume, wherein contain 20%Nu-serum (Collaborative Reserach, Bedford, MA), the DEAE-dextran of 300 mcg/ml, 200 μ M chloroquines are cloned in the LB373-MEL cDNA storehouse of the 100ng among the pcDNAI/Amp, and 50ng be cloned in HLA-A2 among the pcDNAI/Amp.After 4 hours, remove substratum 37 ℃ of cultivations, change the PBS that contains 10% methyl-sulphoxide (DMSO) with 50 microlitres.After 2 minutes, remove this substratum, change the DMEM that contains 10%FCS with 200 microlitres.

After changing the substratum that is over, the HOB cell was cultivated 48 hours at 37 ℃.Then with CTL 246/76 screening transfection thing.After for the first time removing substratum, in each aperture, add 2000 CTL 246/76 cells in the substratum of the IL-2 that contains 25U/ml that is present in 100 microlitres.Then, be present in TNF quantity in the supernatant by measuring cytotoxicity measurement to WEHI 164.13 cells.TNF content in most Xiao Chi all is lower than 5pg/ml.The cDNA that all shows higher concentration in double Xiao Chi is cloned in bacterium.Extract their plasmid DNA, transfection is in the HOB cell that has HLA-A2.With CTL246/76 screening transfection thing.A cDNA clone has provided the TNF of the high density of being produced by CTL246/76.This cDNA is measured sequence, compare, determined that it is the brain glycogen phosphorylase of encoding with the dna sequence data storehouse.

Embodiment 3

To determining of codes for tumor rejection antigen part in the brain glycogen phosphorylase

According to routine techniques, carry out enzyme and cut the fragment for preparing brain glycogen phosphorylase cDNA with the 3 ' end of exonuclease from cDNA, be cloned in the expression vector then, and transfection is in the above-mentioned HOB cell that has HLA-A2.As positive control, with brain glycogen phosphorylase cDNA and HLA-A2 cotransfection in the HOB cell.These transfection things are used to excite the release of CTL 246/76 cell to TNF.Short-movie section with the antigen presentation that can confirm other CTL 246/76 identification of exonuclease preparation is to stop (No. 1 sequence) from its beginning codon to the 100 base pairs.

Produced shorter fragment with PCR.There is not antigen expressed in the 161st position terminated fragment (No. 2 sequence).There is not antigen expressed in the shorter fragment of the 140th position terminated (No. 3 sequence) yet.Therefore, it is important at lucky propylhomoserin (valine) residue of the 141st, 142,143 positions of brain glycogen phosphorylase CTL 246/76 being discerned the brain glycogen phosphorylase Tumor rejection antigen effectively.

Embodiment 4

The evaluation of brain glycogen phosphorylase tumor rejection antigenic peptide

Be synthesized corresponding to 3 ' of No. 2 sequence terminal peptide, and measure its molten born of the same parents the HLA-A2 express cell.In these detect, used the T2 cell.The T2 cell is the HLA-A2 cell with antigen manufacturing deficiency, and their ability that exogenous peptide is provided has been enhanced.The T2 cell with mix mutually corresponding to 3 ' of No. 3 sequence terminal synthetic peptide.Add CTL 246/76 cell, measure molten born of the same parents (Fig. 1) after 4 hours.VB1 peptide (GLAGLGDVAEVRKSFNR, No. 4 sequence) has stimulated the molten born of the same parents of the T2 cell that has HLA-A2 effectively.In order to determine the border of brain glycogen phosphorylase Tumor rejection antigen, we are to a series of 17mer peptide (No. 4 sequence) deutero-10mer, 11mer peptide (5-11 sequence) from being used for stimulating the molten born of the same parents of CTL 246/76 cell in the past, promptly have 10 or 11 and amino acid whose peptide, carried out measuring (Fig. 1).One (LGDVAEVRKS, No. 8 sequence) in them discerned by CTL 246/76, and still, its degree can not show a candle to VB1 peptide (No. 4 sequence), and this just advises that it is important amino acid to CTL 246/76 effective recognition that 9 peptides (No. 8 sequence) lack one.The 10mer peptide (GLGDVAEVRK, No. 7 sequence) that at once is connected to glycine in the terminal of No. 8 sequence just can be discerned effectively by CTL 246/76, to the peptide of No. 5 and No. 9 sequence too.

Embodiment 5

The activity of brain glycogen phosphorylase tumor rejection antigenic peptide

This example has shown the effect of stimulation of brain glycogen phosphorylase TRA peptide to the molten born of the same parents of HLA-A2 express cell, and 6 peptides, 7 peptides, 8 peptides, 9 peptides, 10 peptides and 11 peptides efficient separately.

Remove an amino acid seriatim from the amino acid terminal of 11 peptide VB2 (No. 5 sequence), just prepared the brain glycogen phosphorylase peptide that size has been reduced.These peptides have 10,9,8,7 or 6 amino acid respectively, represent 12-16 sequence (see figure 2) respectively.To these peptides with the test of dose response detection methods they by CTL 246/76 cell to HLA-A2 ⁺The molten born of the same parents' of T2 cell initiating activity.Freeze dried peptide is dissolved among the DMSO with 20mg/ml, and the acetate with 10mM is diluted to 2mg/ml then, and is kept at-80 ℃.To target cell, i.e. HLA-A2 ⁺The T2 cell is used ⁵¹The Cr mark, cultivation is after 1 hour down at 37 ℃, and fully cleaning is to remove unconjugated marker.With anti-HLA-A2 antibody, be MA2.1 (W  lfel et al., EuropeanJournal of Immunology 24:759-764,1994) the T2 cell is carried out pre-treatment (Fig. 2 A) or do not carry out pre-treatment (Fig. 2 B), in 96 hole droplet dishes, cultivate then, add the peptide of different concns, cultivated 30 minutes down for 37 ℃.Then, add CTL 246/76 with equal-volume, the ratio of its effector and target is 30: 1.Measure after 4 hours ⁵¹The release of Cr.Fig. 2 has provided the result of these detection reaction.To having the T2 cell of HLA-A2,8mer, 9mer and 10mer peptide (12-14 sequence) have showed the most effective molten born of the same parents to stimulate.Low order of magnitude of peptide that the specific activity of the peptide of 11mer (VB1, No. 5 sequence) is best.Low two orders of magnitude of peptide that the specific activity of the peptide of 7mer (No. 15 sequence) is best.The peptide of 6mer (No. 16 sequence) does not show activity or the very little activity of performance.

Embodiment 6

The brain glycogen phosphorylase expression of gene

Carry out the expression that PCR measures brain glycogen phosphorylase, the primer of use is as follows: No. 17 sequence:

No. 18 sequence of 5 '-TGC CAG GCA CAG GTG GAC CA-3 ' (meaningful primer, Nucleotide 2369-2388):

5 '-CAG ACC CCA GAA TCC AGA GGC-3 ' (meaningless primer, Nucleotide 2890-2910)

At first, whole RNA are extracted from concrete sample with known technology.Prepare cDNA with this RNA.The program of preparation cDNA comprises, merges the 5x reversed transcriptive enzyme damping fluid of 4 microlitres, the various dNTP (10mM) of 1 microlitre, dithiothreitol (DTT), (100mM), the dT-15 primer (20 μ M) of 2 microlitres, the RNasin (40 units/μ l) of 0.5 μ l and the M-MLV reversed transcriptive enzyme (200 units/μ l) of 1 μ l of 2 microlitres.Then, the RNA template (1 μ g/3.25 μ l water, or the total RNA template of 2 μ g) that adds 6.5 μ l.The cumulative volume of this mixture is 20 μ l.Under 42 ℃, mix and cultivated 60 minutes, then in cooled on ice.The water that adds 80 μ l again makes cumulative volume reach 100 μ l.This mixture is kept under-20 ℃ until being used in PCR.

The reagent that carries out PCR comprises:

The 10xDynaZyme damping fluid of 5 μ l

The various primers of 20pmole

The various dNTP of 5nanomole

The polysaccharase of 1 unit " Dynazyme " (2 units/μ l)

The cDNA of 5 μ l (corresponding to total RNA of 100ng)

Water, making final volume is 50 μ l

Merge these materials, add 1 mineral oil reservoir then.This mixture is transferred in the thermo cycler, be preheating to 94 ℃, carried out a round-robin in following 15 minutes with 94 ℃ then and amplify, carry out 25 circulations subsequently, specific as follows:

1 minute, 94 ℃

30 seconds, 65 ℃

2 minutes, 72 ℃ last 72 ℃ of expansions of carrying out 15 minutes.Containing upward demonstration PCR product of the sepharose of ethidium bromide (1.5%).

The brain glycogen phosphorylase gene has showed the overexpression in tumour.The expression level of finding in this expression of gene level experiment all low (Fig. 3) than the LB373-MEL cell that in all healthy tissuess, carries out.Particularly, the expression of this gene in normal suprarenal gland, bladder, breast, colon, uterine endometrium, heart, kidney, liver, myometrium, ovary, retina, spleen, stomach and testis is all very weak.But this gene all obtains expressing (Fig. 3) in various neoplasmic tissue sample.10 times of expression have all been found in the melanoma 15% or the colon of similar ratio or ovary or the retina cancer to the brain glycogen phosphorylase more than 40 times.These results are confirmed by the dyeing to tumor sample, with being that specific antiserum(antisera) (Ignacio et al., BrainRes.529:42-49,1990) dyes to brain glycogen phosphorylase, tumor tissues obtains highly coloured, and healthy tissues on every side is not colored.

Embodiment 7

Liver or muscle odd-shaped homeopeptide are not discerned by CTL 246/76

The abnormity of the liver of glycogen phosphorylase, muscle and brain has shown about 80% amino acid whose homogeny (Newgard et al., J.Biol.Chem.263:3850-3857,1988).In order to prove that Tumor rejection antigen is that abnormity to brain is specific, the 11 peptide abnormity (GIVGVENVAEL that synthesized liver, No. 19 sequence) and 11 peptide abnormity (GLAGVENVIEL of muscle, and be used for the dose response that the chromium described in the foregoing description 5 discharges and detect No. 20 sequence).As shown in Figure 4, the peptide of liver and muscle does not excite molten born of the same parents, and the brain peptide, promptly VB1 (No. 5 sequence) then causes the molten born of the same parents of specificity.

Embodiment 8

CTL 246/76 is not to the molten born of the same parents of normal cell

This example has been described the molten born of the same parents to the CTL of various clones, comprise with or peptide that need not No. 14 sequence cultivate.In dose response detects, carry out molten born of the same parents' detection with 246/76 pair of LB373-MEL cell of CTL, the normal B cell of use the patient LB373 of EBV (LB373-EBV) conversion, same patient's normal circumference blood lymphocyte (LB373-PBL).These cells are cultivated with CTL 246/76, and shown in Fig. 5 A, these molten born of the same parents detect as mentioned above an effector to the target ratio.The molten born of the same parents that CTL 246/76 only produces the LB373-MEL cell show that LB373-EBV and LB373-PBL cell do not discerned by CTL, because these cells are not generally expressed the brain glycogen phosphorylase Tumor rejection antigen.

What next determine is, if with the auxiliary words of brain glycogen phosphorylase peptide, whether these cells can be by the molten born of the same parents of CTL so.In detecting according to the dose response described in the embodiment of front, the peptide that has detected No. 14 sequence to CTL246/76 at LB373-MEL cell, LB373-EBV cell, LB373-PBL cell, HLA-A2 ⁺The molten born of the same parents' that produce in the T2 cell initiating activity.Fig. 5 B has shown the result that these dose responses detect.LB373-EBV cell and LB373-PBL cell be by CTL 246/76 molten born of the same parents, and still, non-clone T2 from body is but by CTL 246/76 molten born of the same parents.

The invention still further relates to the improper expression of people's brain glycogen phosphorylase.The gene of coding people brain glycogen phosphorylase provides in No. 21 sequence.Various allelotrope all are parts of the present invention.These allelotrope have with No. 21 sequence greater than 95% homology and coding brain glycogen phosphorylase tumor rejection antigen precursor.Under stringent condition, they hybridize in the nucleic acid molecule that contains No. 21 sequence.Term " stringent condition " refers to conditional parameter known in the art in this article.The various parameters of nucleic acid hybridization can be referring to the document of those these methods that collected, for example, MolecularCloning:A Laboratory Manual.J.Sambrook.et al., eds., SecondEdition.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; Or Current Protocols in Molecular Biology, F.M.Ausubel, et al., eds., John Wiley ﹠amp; Sons, Inc., New York.More specifically, stringent condition refers to the hybridization that following condition is carried out in this article, and 65 ℃, hybridization buffer is 3.5 * SSC, 0.02%Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 25mM NaH ₂PO ₄(pH 7), 0.5%SDS, 2mM EDTA.SSC is the Trisodium Citrate of sodium-chlor/0.15M of 0.15M, pH7; SDS is a sodium lauryl sulphate; EDTA is an ethylene diaminetetraacetic acid.After the hybridization, the film that has been transferred DNA is at room temperature washed with 2 * SSC, washes with 0.1 * SSC/0.1 * SDS at 65 ℃ then.

Certainly, can also use other condition, reagent etc., as long as reach same stringent condition.Those skilled in the art is familiar with these conditions, therefore need not to give unnecessary details.It should be understood that those skilled in the art can change these conditions, so that clearly determine the homologue and the allelotrope of nucleic acid molecule of the present invention.Those skilled in the art also know the method for screening cell, particularly screen cancer cells method, express the gene pool of these molecules etc., and separate the method for relevant nucleic acid molecule and measure the method for the sequence of these nucleic acid molecule.

The improper expression of brain glycogen phosphorylase can detect with several different methods.For example, having described brain glycogen phosphorylase in some documents is specific antibody, and these antibody can also prepare by ordinary method, and wherein some are given detailed description following.Be more preferably, detect expression (and the relative expression's level between the various tissue) by measuring mRNA.For example, the expression of brain glycogen phosphorylase in tumour cell and healthy tissues can compare with control cells or homologous tissue.For reaching this purpose, can adopt PCR and other technology.The brain glycogen phosphorylase Auele Specific Primer can be used for any one couple of PCR primers to set up and to arrange optionally to amplify the brain glycogen phosphorylase gene.Such Auele Specific Primer will be hybridized with the Nucleotide total length that only exists only in the brain glycogen phosphorylase, but to the then only hybridization of part of non-brain glycogen phosphorylase gene.For effective PCR primer and to the evaluation of brain glycogen phosphorylase, then the foundation of the Auele Specific Primer of brain glycogen phosphorylase and arranging make its with the glycogen phosphorylase of non-brain in the hybridization effectively of 3 ' end.Not matching of 3 ' end of primer make its with other glycogen phosphorylase when hybridization of non-brain, can get rid of effective amplification to these genes.Those skilled in the art can according to the glycogen phosphorylase odd-shaped sequence of disclosed brain, liver and muscle select primer (referring to, Newgard et al., J.Biol.Chem.263:3850-3857,1988).To those skilled in the art, other the method that can distinguish homology of nucleotide sequence all is conspicuous as the chain reaction methods such as (" LCR ") of ligase enzyme.

The present invention also comprises the application to the nucleotide sequence that has comprised alternative codon, and alternative codon coding wherein is by the identical amino-acid residue of brain glycogen phosphorylase coded by said gene.For example, described in the above embodiments 5 like that, decapeptide LAGLGDVAEV (No. 12 sequence) is the brain glycogen phosphorylase Tumor rejection antigen.Leucine residue (the 1st, 4 amino acid of No. 12 sequence) is respectively by codon CTG and CTA coding.Except CTG and CTA, leucine residue can be by codon CTC, CTT, TTA and TTG coding.In these six kinds of codons any one is being equivalent aspect the coding leucine residue.Therefore, for it is apparent that, any one leucine coding nucleotide triplet can be used in the device that instructs protein synthesis for those skilled in the art, comes in vivo or external integration leucine residue.Similarly, the nucleotide triplet of other amino-acid residue comprises in the composition of coding brain glycogen phosphorylase Tumor rejection antigen: CGA, CGC, CGG, CGT, AGA and AGG (arginine codon); GGA, GGC, GGG and GGT (codon glycine); GCA, GCC, GCG and GCT (L-Ala codon); GAC and GAU (aspartic acid codon); CGA, CGC, CGG, CGT, AGA and AGG (arginine codon).Other amino-acid residue can have a plurality of nucleotide sequences to encode similarly.Therefore, the present invention includes because the degeneracy of genetic code and the nucleic acid separated with biology are different from the nucleic acid of degeneracy on the codon sequence.

The above embodiments also show the separation to brain glycogen phosphorylase TRAs peptide.These peptides are translation products (No. 21 sequence) of the nucleic acid of brain glycogen phosphorylase.Therefore, those skilled in the art just knows, those translation products that form finally is provided that are used for producing brain glycogen phosphorylase TRA can be the sequences of any length, as long as they include the core of the brain glycogen phosphorylase TRA of No. 15 sequence representative.Shown in the above embodiments, little of 7,8,9,10 or 11 amino acid whose peptides, and big can in needs, suitably be processed to by brain glycogen phosphorylase cDNA amino acid sequence coded, provide and by CTL 246/76 identification by HLA-A2.The peptide of No. 15 sequence can add 1,2,3,4,5,6,7,8,9,10 or more a plurality of amino acid in its one or both ends.Therefore, Tumor rejection antigen can comprise 7 successive amino acid and No. 15 sequence of No. 21 sequence, 8 successive amino acid and No. 15 sequence of comprising No. 21 sequence, 9 successive amino acid and No. 15 sequence of comprising No. 21 sequence, 10 successive amino acid and No. 15 sequence of comprising No. 21 sequence, 11 successive amino acid and No. 15 sequence of comprising No. 21 sequence, 12 successive amino acid and No. 15 sequence of comprising No. 21 sequence, 13 successive amino acid and No. 15 sequence of comprising No. 21 sequence, 14 successive amino acid and No. 15 sequence of comprising No. 21 sequence, 15 successive amino acid and No. 15 sequence of comprising No. 21 sequence, 16 successive amino acid and No. 15 sequence of comprising No. 21 sequence, 17 successive amino acid and No. 15 sequence of comprising No. 21 sequence, 18 successive amino acid and No. 15 sequence of comprising No. 21 sequence, comprise 19 successive amino acid and No. 15 sequence of No. 21 sequence, and/or comprise 100 successive amino acid and No. 15 sequence to No. 21 sequence.The antigen part of these peptides is provided divided by the class by HLA in the physiological condition incision.

The aminoacid sequence of the protein of brain glycogen phosphorylase TRAs or peptide of deriving can be natural or the non-natural source, be that they can comprise natural brain glycogen phosphorylase TRAP molecule, or comprise adorned sequence, as long as aminoacid sequence has kept when occurring the Tumor rejection antigen sequence that can be discerned by CTL on cell surface.For example, the brain glycogen phosphorylase Tumor rejection antigen of this moment can be a fusion rotein, comprise brain glycogen phosphorylase Tumor rejection antigen and incoherent aminoacid sequence, at No. 15, No. 14, No. 13, No. 12, or the synthetic peptide of the aminoacid sequence shown in No. 5 sequence, the peptide of mark, the peptide of from melanoma patient, separating, the peptide of from the cell culture of expressing brain glycogen phosphorylase, separating, be paired to the peptide on the non-peptide molecule, for example, in the some drugs transfer system those, and other the molecule that comprises No. 15 aminoacid sequence.

From these embodiment, it can also be seen that, the present invention includes the application of these sequences in expression vector, and the host cell of transfection and clone, comprise prokaryotic cell prokaryocyte (for example intestinal bacteria) and eukaryotic cell (for example, Chinese hamster ovary celI, COS cell, HeLa cell, yeast expression system and the recombinant baculovirus expression in insect cell).What expression vector required is but that those above-mentioned relevant sequences are connected on the promotor by the place of working.As found, people HLA-A2 provides by these gene deutero-TRA, and expression vector can also comprise the particularly nucleotide sequence of HLA-A2 molecule of coding HLA molecule.When carrier contains these two kinds of encoding sequences, just can use it for and those not expressed generally any one cell carries out transfection in them.The encoding sequence of TRAP or TRA can use separately, for example, and when host cell is expressed HLA-A2.Certainly, to the not special restriction of operable host cell.Can be used for HLA-A2 when needed as the carrier that contains two kinds of encoding sequences provides the cell, and the nucleic acid of coding TRAP or TRA can be used in the host cell of not expressing HLA-A2.

The present invention also comprises so-called expression test kit, and these test kits can be that those skilled in the art prepares needed expression vector.Such test kit comprises at least two kinds of above-mentioned materials and placed apart.The composition that can also add if desired, other.

Invention described herein has multiple application, and wherein some are as described below.At first, the present invention can diagnose those skilled in the art to the disease that is expressed as feature with TRAP.These diagnostic methods comprise determine the TRAP gene and/or from the expression of its deutero-TRAs the TRA that provides by HLA-A2.When definite TRAP expression of gene, can finish by the nucleic acid detection method of any routine, comprise polymerase chain reaction, and with the detection method of the hybridization probe of mark.Under latter event, to the detection of the binding partner of the complex body of TRA and HLA, for example detection of antagonist then is preferred.Another method is that TNF discharges detection, and is just as described above.

The TRAPs or the TRAs (those skilled in the art has known numerous this class HLA molecules, and these molecules include but not limited to by those molecules of HLA-A, HAL-B, HLA-C, HLA-E, HLA-F and HLA-G genes encoding) that utilize method described here can also separate that encode by brain glycogen phosphorylase, that discerned by other ctl clone of other and/or be provided by other HLA molecule.The method of the more known distortion of those skilled in the art can be used to obtain separated TRAP molecule, and/or by its deutero-TRAs.Protein can purify from its natural production cell.In addition, expression vector can be incorporated into and cause proteinic production in the cell.In another approach, can utilize the method for microinjection or other method mRNA to be incorporated into the proteinic production that causes in the cell coded.In not celliferous extract,, also can be used to produce protein as in the molten born of the same parents' system system of reticulocyte, translating mRNA.The peptide that contains TRAs of the present invention can also be external synthetic.Those skilled in the art can adopt the method for knowing to come isolated protein, to obtain separated TRAP and/or by its deutero-TRAs.These methods include but not limited to immune chromatograph, HPLC, size exclusion chromatogram, ion-exchange chromatography and immune affinity chromatographic etc.These separated molecules are in processed back and be used as TRA or when providing as the complex body of TRA and HLA such as HLA-A2, and can combining with materials such as vehicle, to produce treatment be that the disease of feature is useful vaccine with expression TRAP molecule.In addition, from TRA/HAL complex body cell is provided on its surface, for example can prepare vaccine in the transfectional cell of the cancer cells of non-breeding, non-breeding.All with the situation of cell as vaccine under, these cells can be that using exciting ctl response is a kind of or whole encoding sequence cells transfected in the necessary component, or have expressed these two kinds of molecules and do not need cells transfected.Vaccine also comprises exposed DNA or RNA, their coding brain glycogen phosphorylase TRA or its precursors, can be in external generation, suck and other mode administration by injection, partickle bombardment, nasal cavity.The vaccine of " exposed nucleic acid " type has shown immune response stimulating, comprises that to the exposed coded peptide of nucleic acid be the generation (Science 259:1745-1748,1993) of specific CTLs.

The complex body of TRAP molecule, relative TRAs and TRA and HLA may be used to produce antibody, and the method that needs all is known in the art.The principle of antibody producing commonly used with job requirement etc., can be referring to Catty, D., Antibodies, A Practical Approach, Vol.1.IRL Press, WashingtonDC (1988); Klein J., Immunology:The Science of Cell-Non-CellDiscrimination, John Wiley and Sons, New York (1982); Kennett, R., et al., Monoclonal Antibodies.Hybridoma.A New Dimension InBiological Analyses.Plenum Press, New York (1980); Campbell, A., Monoclonal Antibody Technology, in Laboratory Techniques andBiochemistry and Molecular Biology, Vol.13 (Burdon.R.et al., EDS) .Elsevier Amsterdam (1984); And Eisen, H.N., Microbiology, third edition.Davis.B.D.et al., EDS. (Harper ﹠amp; Rowe, Philadelphia (1980).

Therefore, antibody of the present invention can comprise that providing protein, proteinic fragment, marking protein or its segmental cell to wait to animal produces polyclonal antibody with any method preparation.The production method of monoclonal antibody is well-known to those skilled in the art.As described herein, these antibody can be used to identify the tissue or the protein purification of marking protein.These antibody can also combine with other preparation, and for example, specific show tags thing or anticancer preparation include but not limited to methotrexate, radioiodine compound, toxin, for example ricin, or other cytostatic medicament or the molten born of the same parents' medicine of cell etc.Antibody prepared in accordance with the present invention is specific antibody to described TRA/HLA complex body preferably.

In this article, term " disease " refers to the pathological conditions of having expressed Tumor rejection antigen.One of example of these diseases is a cancer, and is concrete as melanoma.

Methods of treatment more according to the present invention can allow patient's immune system react, and cause the cell that TRA is provided such as the molten born of the same parents of HLA-A2 cell.One of these methods are that the patient to the improper cell with described phenotype provides complex body is specific from body CTLs.At the such CTLs of external development is within those skilled in the art's the knowledge category.Generally speaking, take cell sample on one's body from the patient earlier, for example red blood cell allows these cells contact with the cell that provides complex body and can excite CTLs to breed then.The target cell can be transfected, for example the HeLa cell of the above type.These cells transfected provide needed complex body on its surface, and with after corresponding C TLs combines, can stimulate its breeding.The HeLa cell, for example described herein those, all generally can obtain as other host cell.Selectivity production to ctl clone has given description in the above.Can offer the patient from body CTLs with what expanded by cloning.Other be that specific CTLs also can separate and offer the patient with method similarly to brain glycogen phosphorylase.

Concrete methods of treatment, as be called adoptive transfer [Greenberg.J.Immunol.136 (5): 1917 (1986); Riddle et al., Science 257:238 (7-10-92); Lynchet al., Eur.J.Immunol.21:1403-1410 (1991); Kast et al., Cell59:603-614 (11-17-89)] method, be to provide the cell of required complex body to combine, to cause the breeding of specific CTLs with CTLs.CTLs with breeding offers the patient with cellular abnormality then, and cellular abnormality wherein is that to provide the abnormal cells of special complex body with some be feature.Then, abnormal cells is carried out molten born of the same parents, thereby obtain desired result of treatment with CTLs.

Above-mentioned treatment has supposed that some patient's cellular abnormality provides relevant HLA/TRA complex body at least.This point can easily be determined, because those skilled in the art very has been familiar with identifying the method for the cell that the specific HLA molecule is provided, and the method for identifying the cell of the DNA that expresses relevant sequence, be the brain glycogen phosphorylase sequence at this.In case identified the cell that related complexes is provided with aforesaid screening method, just they can have been contacted with the sample that contains CTLs from the patient.If this blended CTL sample has carried out molten born of the same parents to the cell that complex body is provided, so just can suppose to be provided, and the patient is the proper object with above-mentioned method treatment from TRA deutero-brain glycogen phosphorylase.

The adoptive transfer method is not according to methods of treatment of the present invention form only to be arranged.Use some other method, can also excite CTLs in vivo.One of such method is the non-reproductive ability cell that complex body is expressed in the use described in the above.Employed in the method cell can be those general cells of expressing complex body, for example, through the radiating tumour cell, or to use for complex body is provided be one or two cells transfected " referring to Chen et al.; Proc.Natl.Acad.Sci.USA88:110-114 (January, 1991) " in the necessary gene.This piece document illustration this method, illustrated in methods of treatment and to have used the transfectional cell of expressing the HPVE7 peptide.Operable cell category is a lot.Similarly, the carrier that contains one or two significant gene also can be used.Preferably viral or bacillary carrier.For example, the nucleic acid of coding brain glycogen phosphorylase TRA can be connected at some cell with in organizing with operability ground and be instructed on brain glycogen phosphorylase TRA expression promoter sequence or the enhancer sequence.Nucleic acid can be incorporated in the expression vector.Expression vector can be the extrachromosomal nucleic acid of unmodified, plasmid or the viral genome that can insert exogenous nucleic acid such as those coding brain glycogen phosphorylases TRAs foundation or that modify.The nucleic acid of coding brain glycogen phosphorylase TRA can also be inserted in the retroviral gene group, thereby promotes the integration of nucleic acid in the genome of target tissue or cell.In these systems, significant gene is carried by microorganism, and for example, vaccine virus, retroviral, bacterium BCG and those " infect " material of host cell.These can provide the cell of significant complex body, and just can be bred from the cell of body CTLs identification etc.

TRAP or its pungency fragment are combined with promote the adjuvant that inserts in the body of the cell that HLA-A2 is provided, also can reach similar effects.TRAP is processed, obtain the coordination peptide of HLA molecule, TRA then is provided with the form that does not need further processing.Generally speaking, patient brain glycogen phosphorylase TRAP and/or its TRAs of effective dose that can get an injection under the skin.Booster dose can and then be used in the dosage back of beginning, carries out immunity according to routine techniques then.

As the part of immunization method, those have the material that causes immunoreactive potentiality can offer the patient together with the peptide of nucleic acid or cancer vaccine.These compounds with immune response potentiality can be divided into adjuvant and cytokine.Adjuvant by antigenic storehouse (extracellular or have a liking for cell in huge) is provided, activation is huge has a liking for cell and stimulate special lymphocyte to improve immune response.The known adjuvant of ability has multiple, concrete example comprises MPL (SmithKline Beecham), it is by the congener behind purification and the acidic hydrolysis in Salmonellas (the Salmonellaminnesota Re 595) lipopolysaccharides, QS21 (SmithKline Beecham), the i.e. QA-21 saponin(e that purifying comes out from Quillja saponaria extract, and with the various water in oil emulsion of biodegradable oil such as shark alkene and/or tocopherol preparation.Cytokine is because it is to lymphocytic hormesis but can be used for immunization method.The cytokine that can reach this purpose that those skilled in the art knows most comprises having shown the interleukin 12 (IL-12) (Science 268:1432-1434,1995) that improves vaccine protection effect.

When administration, the form that therapeutic composition of the present invention can be prepared in the acceptable preparation of pharmacology is come administration.This pharmaceutical preparation can contain the salt, buffer reagent, sanitas, consistency carrier of pharmacology acceptable concentration, additional immune potentiality preparation such as adjuvant and cytokine, and the medicament that contains or do not contain other.

The dosage of pharmaceutical preparation of the present invention is an effective dose.Effective dose refers to and can rely on self or produce needed immunoreactive dosage with other further dosage.In the treatment cancer, needed reaction is the development that suppresses cancer.This can be temporarily to slow down advancing of disease, but more preferably permanently stops advancing of disease.Can monitor advancing of disease according to diagnostic method of the present invention or conventional monitoring method.

When needs use medicinal compositions of the present invention to stimulate to obtain needed immune response, may relate to and stimulate humoral antibody to react the antibody titer that improves serum, pair cell toxicity lymphocyte carries out the cloning expansion, or relates to other needed immune response.It is believed that the effective dose that just can become immune response stimulating or inhibition cancer development in the immunogen of 1 nanogram(ng)/kilogram-100 milligram/kilogram dosage range according to the mode of administration.The preferred dosage scope is 500 nanogram(ng)s-500 microgram/kilogram.Concrete dosage will come final decision according to numerous factors, comprise the number of times of medicament categories, administration, patient's situation, as degree of age, state of health, height, body weight, disease etc.These factors all are that those skilled in the art is known, can not transnormal test and determine.

Other all respects of the present invention will be understood those skilled in the art, therefore need not to give unnecessary details.

Term used herein all is illustrative, from any aspect restriction the present invention, does not also get rid of any equivalent, it should be understood that the present invention also has numerous modification.These modification should not be considered to break away from spirit of the present invention and invention scope.And these modification all are that the scope of claims of the present invention is included.

Be the information of various sequences of the present invention below.1. overall information

(ⅰ) applicant:

(A) title: LUDWIG cancer research institute

(B) street:

(C) city: Zurich

(E) country: Switzerland

(F) postcode:

(ⅰ) applicant:

(A) title: University of California's trustship council

(B) street: No. 300, lakeside road, the 22nd layer

(C) city: Auckland city

(E) state: California

(F) postcode: 94612-3550

(ⅱ) denomination of invention: brain glycogen phosphorylase cancer antigen

(ⅲ) sequence number: 25

(ⅳ) address:

(A) address: Wolf, Green's Fu Erde ﹠amp; Sai Kesi, P.C.

(B) street: No. 600, main road, Atlanta

(C) city: classic city

(D) state: Massachusetts

(E) country: the U.S.

(F) postcode: 02110

(ⅴ) computer reads form:

(A) type of device: floppy disk

(B) computer: ibm compatible personal computer machine

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, Version#1.25

(ⅵ) existing application information:

(A) application number:

(B) applying date:

(C) classification number:

(ⅶ) priority application information:

(A) application number: 08/672,351

(B) applying date: on June 25th, 1996

(ⅷ) proxy/agency's information:

(A) title: VAN AMSTERDAM, JOHN R.

(B) number of registration: 40,212

(C) reel number: L0461/7004WO

(ⅸ) phone information:

(A) phone: 617-720-3500

(B) fax: the information of No. 1 sequence of 617-720-2441:

(ⅰ) sequence signature

(A) length: 110 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topology: linearity

(ⅱ) molecule type: cDNA

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: N-terminal

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) description of sequence: the information of No. 2 sequence of No. 1 sequence A TGGGCGAAC CGCTGACGGA CAGCGAGAAG CGGAAGCAGA TCAGCGTGCG CGGCCTGGCG 60GGGCTAGGCG ACGTGGCCGA GGTGCGGAAG AGCTTCAACC GGCACTTGCA 110

(ⅰ) sequence signature

(A) length: 102 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topology: linearity

(ⅱ) molecule type: cDNA

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: N-terminal

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: the information of No. 3 sequence of No. 2 sequence A TGGGCGAAC CGCTGACGGA CAGCGAGAAG CGGAAGCAGA TCAGCGTGCG CGGCCTGGCG 60GGGCTAGGCG ACGTGGCCGA GGTGCGGAAG AGCTTCAACCGG 102

(ⅰ) sequence signature

(A) length: 81 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topology: linearity

(ⅱ) molecule type: eDNA

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: N-terminal

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: the information of No. 4 sequence of No. 3 sequence A TGGGCGAAC CGCTGACGGA CAGCGAGAAG CGGAAGCAGA TCAGCGTGCG CGGCCTGGCG 60GGGCTAGGCG ACGTGGCCGA G 81

(ⅰ) sequence signature

(A) length: 17 amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅰ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅷ) sequence description: the information of 15 10 15 No. 5 sequence of No. 4 sequence Gly Leu Ala Gly Leu Gly Asp Val Ala Glu Val Arg Lys Ser Phe Asn Arg

(ⅰ) sequence signature

(A) length: 11 amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 5 sequence

Gly?Leu?Ala?Gly?Leu?Gly?Asp?Val?Ala?Glu?Val

The information of 15 10 No. 6 sequence

(ⅰ) sequence signature

(A) length: ten amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 6 sequence

Ala?Gly?Leu?Gly?Asp?Val?Ala?Glu?Val?Arg

The information of 15 10 No. 7 sequence

(ⅰ) sequence signature

(A) length: ten amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 7 sequence

Gly?Leu?Gly?Asp?Val?Ala?Glu?Val?Arg?Lys

The information of 15 10 No. 8 sequence

(ⅰ) sequence signature

(A) length: ten amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 8 sequence

Leu?Gly?Asp?Val?Ala?Glu?Val?Arg?Lys?Ser

The information of 15 10 No. 9 sequence

(ⅰ) sequence signature

(A) length: ten amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 9 sequence

Gly?Asp?Val?Ala?Glu?Val?Arg?Lys?Ser?Phe

The information of 15 10 No. 10 sequence

(ⅰ) sequence signature

(A) length: ten amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 10 sequence

Asp?Val?Ala?Glu?Val?Arg?Lys?Ser?Phe?Asn

The information of 15 10 o.11 sequences

(ⅰ) sequence signature

(A) length: ten amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: o.11 sequence

Val?Ala?Glu?Val?Arg?Lys?Ser?Phe?Asn?Arg

The information of 15 10 No. 12 sequence

(ⅰ) sequence signature

(A) length: ten amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅰ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 12 sequence

Leu?Ala?Gly?Leu?Gly?Asp?Val?Ala?Glu?Val

The information of 15 10 No. 13 sequence

(ⅰ) sequence signature

(A) length: nine amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 13 sequence

Ala?Gly?Leu?Gly?Asp?Val?Ala?Glu?Val

The information of 15 No. 14 sequence

(ⅰ) sequence signature

(A) length: eight amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅶ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 14 sequence

Gly?Leu?Gly?Asp?Val?Ala?Glu?Val

The information of 15 No. 15 sequence

(ⅰ) sequence signature

(A) length: 7 amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: the 1 No. 5 sequence

Leu?Gly?Asp?Val?Ala?Glu?Val

The information of 15 No. 16 sequence

(ⅰ) sequence signature

(A) length: six amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 16 sequence

Gly?Asp?Val?Ala?Glu?Val

The information of 15 No. 17 sequence

(ⅰ) sequence signature

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: cDNA

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) source:

(A) structure: Homo sapiens

(ⅵ) sequence description: the information of No. 18 sequence of No. 17 sequence TGCCAGGCAC AGGTGGACCA 20

(ⅰ) sequence signature

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: cDNA

(ⅲ) hypothesis: do not have

(ⅳ) antisense: be

(ⅴ) source:

(A) structure: Homo sapiens

(ⅵ) sequence description: the information of No. 19 sequence of No. 18 sequence C AGACCCCAG AATCCAGAGG C 21

(ⅰ) sequence signature

(A) length: 11 amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: the 1 No. 9 sequence

Gly?Ile?Val?Gly?Val?Glu?Asn?Val?Ala?Glu?Leu

The information of 15 10 No. 20 sequence

(ⅰ) sequence signature

(A) length: 11 amino acid

(B) type: amino acid

(C) chain: strand

(D) topology: linearity

(ⅱ) molecule type: peptide

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 20 sequence

Gly?Leu?Ala?Gly?Val?Glu?Asn?Val?Ile?Glu?Leu

The information of 15 10 No. 21 sequence

(ⅰ) sequence signature

(A) length: 4066 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topology: linearity

(ⅱ) molecule type: cDNA

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) source:

(A) structure: Homo sapiens

(ⅵ) feature:

(A) title/key: CDS

(B) position: 35..2566

(ⅶ) sequence description: No. 21 sequence C CTCCATCTC TTTTCCTCCG CCTCCGCCGG CGCG ATG GGC GAA CCG CTG ACG 52

Met?Gly?Glu?Pro?Leu?Thr

1 5GAC?AGC?GAG?AAG?CGG?AAG?CAG?ATC?AGC?GTG?CGC?GGC?CTC?GCG?GGG?CTA 100Asp?Ser?Glu?Lys?Arg?Lys?Gln?Ile?Ser?Val?Arg?Gly?Leu?Ala?Gly?Leu

10 15 20GGC?GAC?GTG?GCC?GAG?GTG?CGG?AAG?AGC?TTC?AAC?CGG?CAC?TTG?CAC?TTC 148Gly?Asp?Val?Ala?Glu?Val?Arg?Lys?Ser?Phe?Asn?Arg?His?Leu?His?Phe

25 30 35ACG?CTG?GTC?AAG?GAC?CGC?AAT?GTG?GCC?ACG?CCC?CGC?GAC?TAC?TTC?TTC 196Thr?Leu?Val?Lys?Asp?Arg?Asn?Val?Ala?Thr?Pro?Arg?Asp?Tyr?Phe?Phe

40 45 50GCG?CTG?GCG?CAC?ACG?GTG?CGC?GAC?CAC?CTC?GTG?GGC?CGC?TGG?ATC?CGC 244Ala?Leu?Ala?His?Thr?Val?Arg?Asp?His?Leu?Val?Gly?Arg?Trp?Ile?Arg?55 60 65 70ACG?CAG?CAG?CAC?TAC?TAC?GAG?CGC?GAC?CCC?AAG?CGC?ATT?TAT?TAT?CTT 292Thr?Gln?Gln?His?Tyr?Tyr?Glu?Arg?Asp?Pro?Lys?Arg?Ile?Tyr?Tyr?Leu

75 80 85TCC?CTG?GAA?TTC?TAC?ATG?GGT?CGC?ACG?CTG?CAG?AAC?ACG?ATG?GTG?AAC 340Ser?Leu?Glu?Phe?Tyr?Met?Gly?Arg?Thr?Leu?Gln?Asn?Thr?Met?Val?Asn

90 95 100CTG?GGC?CTT?CAG?AAT?GCC?TGC?GAT?GAA?GCC?ATC?TAT?CAG?TTG?GCG?TTA 388Leu?Gly?Leu?Gln?Asn?Ala?Cys?Asp?Glu?Ala?Ile?Tyr?Gln?Leu?Gly?Leu

105 110 115GAC?TTG?GAG?GAA?CTC?GAG?GAG?ATA?GAA?GAA?GAT?GCT?GGC?CTT?GGG?AAT 436Asp?Leu?Glu?Glu?Leu?Glu?Glu?Ile?Glu?Glu?Asp?Ala?Gly?Leu?Gly?Asn

120 125 130GGA?GGC?CTG?GGG?AGG?CTG?GCA?GCG?TGT?TTC?CTT?GAC?TCA?ATG?GCT?ACC 484Gly?Gly?Leu?Gly?Arg?Leu?Ala?Ala?Cys?Phe?Leu?Asp?Ser?Met?Ala?Thr135 140 145 150TTG?GGC?CTG?GCA?GCA?TAC?GGC?TAT?GGA?ATC?CGC?TAT?GAA?TTT?GGG?ATT 532Leu?Gly?Leu?Ala?Ala?Tyr?Gly?Tyr?Gly?Ile?Arg?Tyr?Glu?Phe?Gly?Ile

155 160 165TTT?AAC?CAG?AAG?ATT?GTC?AAT?GGC?TGG?GAG?GTA?GAG?GAG?GCC?GAT?GAC 580Phe?Asn?Gln?Lys?Ile?Val?Asn?Gly?Trp?Gln?Val?Gln?Gln?Ala?Asp?Asp

170 175 180TGG?CTG?CGC?TAC?GGC?AAC?CCC?TGG?GAG?AAA?GCG?CGG?CCT?GAG?TAT?ATG 628Trp?Leu?Arg?Tyr?Gly?Asn?Pro?Trp?Gln?Lys?Ala?Arg?Pro?Glu?Tyr?Met

185 190 195CTT?CCC?GTG?CAC?TTC?TAC?GGA?CGC?GTG?GAG?CAC?ACC?CCC?GAG?GGC?GTG 676Leu?Pro?Val?His?Phe?Tyr?Gly?Arg?Val?Glu?His?Thr?Pro?Asp?Gly?Val

200 205 210AAG?TGG?CTG?GAC?ACA?CAG?GTG?GTG?CTG?GCC?ATG?CCC?TAC?GAC?ACC?CCA 724Lys?Trp?Leu?Asp?Thr?Gln?Val?Val?Leu?Ala?Met?Pro?Tyr?Asp?Thr?Pro215 220 225 230GTG?CCC?GGC?TAC?AAG?AAC?AAC?ACC?GTC?AAC?ACC?ATG?CGG?CTG?TGG?TCC 772Val?Pro?Gly?Tyr?Lys?Asn?Asn?Thr?Val?Asn?Thr?Met?Arg?Leu?Trp?Ser

235 240 245GCC?AAG?GCT?CCC?AAC?GAC?TTC?AAG?CTG?CAG?GAC?TTC?AAC?GTG?GGA?GAC 820Ala?Lys?Ala?Pro?Asn?Asp?Phe?Lys?Leu?Gln?Asp?Phe?Asn?Val?Gly?Asp

250 255 260TAC?ATC?GAG?GCG?GTC?CTG?GAC?CGG?AAC?TTG?GCT?GAG?AAC?ATC?TCC?AGG 868Tyr?Ile?Glu?Ala?Val?Leu?Asp?Arg?Asn?Leu?Ala?Glu?Asn?Ile?Ser?Arg

265 270 275GTC?CTG?TAT?CCA?AAT?GAT?AAC?TTC?TTT?GAG?GGG?AAG?GAG?CTG?CGG?CTG 916Val?Leu?Tyr?Pro?Asn?Asp?Asn?Phe?Phe?Glu?Gly?Lys?Glu?Leu?Arg?Leu

280 285 290AAG?CAG?GAG?TAC?TTC?GTG?GTG?GCC?GCC?ACG?CTC?CAG?GAC?ATC?ATC?CGC 964Lys?Gln?Glu?Tyr?Phe?Val?Val?Ala?Ala?Thr?Leu?Gln?Asp?Ile?Ile?Arg295 300 305 310CGC?TTC?AAG?TCG?TCC?AAG?TTC?GGC?TGC?CGG?GAC?CCT?GTG?AGA?ACC?TGT 1012Arg?Phe?Lys?Ser?Ser?Lys?Phe?Gly?Cys?Arg?Asp?Pro?Val?Arg?Thr?Cys

315 320 325TTC?GAG?ACG?TTC?CCA?GAC?AAG?GTG?GCC?ATC?CAG?CTG?AAC?GAC?ACC?CAC 1060Phe?Glu?Thr?Phe?Pro?Asp?Lys?Val?Ala?Ile?Gln?Leu?Asn?Asp?Thr?His

330 335 340CCC?GCC?CTC?TCC?ATC?CCT?GAG?CTC?ATG?CGG?ATC?CTG?GTG?GAC?GTG?GAG 1108Pro?Ala?Leu?Ser?Ile?Pro?Glu?Leu?Met?Arg?Ile?Leu?Val?Asp?Val?Glu

345 350 355AAG?GTG?GAC?TGG?GAC?AAG?GCC?TGG?GAA?ATC?ACG?AAG?AAG?ACC?TGT?GCA 1156Lys?Val?Asp?Trp?Asp?Lys?Ala?Trp?Glu?Ile?Thr?Lys?Lys?Thr?Cys?Ala

360 365 370TAC?ACC?AAC?CAC?ACT?GTG?CTG?CCT?GAG?GCC?TTG?GAG?CGC?TGG?CCC?GTG 1204Tyr?Thr?Asn?His?Thr?Val?Leu?Pro?Glu?Ala?Leu?Glu?Arg?Trp?Pro?Val375 380 385 390TCC?ATG?TTT?GAG?AAG?CTG?CTG?CCG?CGG?CAC?CTG?GAG?ATA?ATC?TAT?GCC 1252Ser?Met?Phe?Glu?Lys?Leu?Leu?Pro?Arg?His?Leu?Glu?Ile?Ile?Tyr?Ala

395 400 405ATC?AAG?CAG?CGG?CAC?CTG?GAC?CAC?GTG?GCC?GCG?CTG?TTT?CCC?GGC?GAT 1300Ile?Asn?Gln?Arg?His?Leu?Asp?His?Val?Ala?Ala?Leu?Phe?Pro?Gly?Asp

410 415 420GTG?GAC?CGC?CTG?CGC?AGG?ATG?TCT?GTG?ATC?GAG?GAG?GGG?GAC?TGC?AAG 1348Val?Asp?Arg?Leu?Arg?Arg?Met?Ser?Val?Ile?Glu?Glu?Gly?Asp?Cys?Lys

425 430 435CGG?ATC?AAC?ATG?GCC?CAC?CTG?TGT?GTG?ATT?GGG?TCC?CAT?GCT?GTC?AAT?1396Arg?Ile?Asn?Met?Ala?His?Leu?Cys?Val?Ile?Gly?Ser?His?Ala?Val?Asn

440 445 450GGT?GTG?GCG?AGG?ATC?CAC?TCG?GAG?ATC?GTG?AAA?CAG?TCG?GTC?TTT?AAG?1444Gly?Val?Ala?Arg?Ile?His?Ser?Glu?Ile?Val?Lys?Gln?Ser?Val?Phe?Lys455 460 465 470GAT?TTT?TAT?GAA?CTG?GAG?CCA?GAG?AAG?TTC?CAG?AAT?AAG?ACC?AAT?GGC?1492Asp?Phe?Tyr?Glu?Leu?Glu?Pro?Glu?Lys?Phe?Gln?Asn?Lys?Thr?Asn?Gly

475 480 485ATC?ACC?CCC?CGC?CGG?TGG?CTG?CTG?CTG?TGC?AAC?CCG?GGG?CTG?GCC?GAT?1540Ile?Thr?Pro?Arg?Arg?Trp?Leu?Leu?Leu?Cys?Asn?Pro?Gly?Leu?Ala?Asp

490 495 500ACC?ATC?GTG?GAG?AAA?ATT?GGG?GAG?GAG?TTC?CTG?ACT?GAC?CTG?AGC?CAG?1588Thr?Ile?Val?Glu?Lys?Ile?Gly?Glu?Glu?Phe?Leu?Thr?Asp?Leu?Ser?Gln

505 510 515CTG?AAG?AAG?CTG?CTG?CCG?CTG?GTC?AGT?GAC?GAG?GTG?TTC?ATC?AGG?GAC?1636Leu?Lys?Lys?Leu?Leu?Pro?Leu?Val?Ser?Asp?Glu?Val?Phe?Ile?Arg?Asp

520 525 530GTG?GCC?AAG?GTC?AAA?CAG?GAG?AAC?AAG?CTC?AAG?TTC?TCG?GCC?TTC?CTG?1684Val?Ala?Lys?Val?Lys?Gln?Glu?Asn?Lys?Leu?Lys?Phe?Ser?Ala?Phe?Leu535 540 545 550GAG?AAG?GAG?TAC?AAG?GTG?AAG?ATC?AAC?CCC?TCC?TCC?ATG?TTC?GAT?GTG?1732Glu?Lys?Glu?Tyr?Lys?Val?Lys?Ile?Asn?Pro?Ser?Ser?Met?Phe?Asp?Val

555 560 565CAT?GTG?AAG?AGG?ATC?CAC?GAG?TAC?AAG?CGG?CAG?CTG?CTC?AAC?TGC?CTG?1780His?Val?Lys?Arg?Ile?His?Glu?Tyr?Lys?Arg?Gln?Leu?Leu?Asn?Cys?Leu

570 575 580CAC?GTC?GTC?ACC?CTG?TAC?AAT?CGA?ATC?AAG?AGA?GAC?CCG?GCC?AAG?GCT?1828His?Val?Val?Thr?Leu?Tyr?Asn?Arg?Ile?Lys?Arg?Asp?Pro?Ala?Lys?Ala

585 590 595TTT?GTG?CCC?AGG?ACT?GTT?ATG?ATT?GGG?GGC?AAG?GCA?GCG?CCC?GGT?TAC?1876Phe?Val?Pro?Arg?Thr?Val?Met?Ile?Gly?Gly?Lys?Ala?Ala?Pro?Gly?Tyr

600 605 610CAC?ATG?GCC?AAG?CTG?ATC?ATC?AAG?TTG?GTC?ACC?TCC?ATC?CGC?GAC?GTC?1924His?Met?Ala?Lys?Leu?Ile?Ile?Lys?Leu?Val?Thr?Ser?Ile?Gly?Asp?Val615 620 625 630GTC?AAT?CAT?GAC?CCA?GTT?GTG?GGT?GAC?AGG?TTG?AAA?GTG?ATC?TTC?CTG?1972Val?Asn?His?Asp?Pro?Val?Val?Gly?Asp?Arg?Leu?Lys?Val?Ile?Phe?Leu

635 640 645GAG?AAC?TAC?CGT?GTG?TCC?TTG?GCT?GAG?AAA?GTG?ATC?CCG?GCC?GCT?GAT?2020Glu?Asn?Tyr?Arg?Val?Ser?Leu?Ala?Glu?Lys?Val?Ile?Pro?Ala?Ala?Asp

650 655 660CTG?TCG?CAG?CAG?ATC?TCC?ACT?GCA?GGC?ACC?GAG?GCC?TCA?GGC?ACA?GGC?2068Leu?Ser?Gln?Gln?Ile?Ser?Thr?Ala?Gly?Thr?Glu?Ala?Ser?Gly?Thr?Gly

665 670 675AAC?ATG?AAG?TTC?ATG?CTC?AAC?GGG?GCC?CTC?ACC?ATC?GGC?ACC?ATG?GAC?2116Asn?Met?Lys?Phe?Met?Leu?Asn?Gly?Ala?Leu?Thr?Ile?Gly?Thr?Met?Asp

680 685 690GGC?GCC?AAC?GTG?GAG?ATG?GCC?GAG?GAG?GCC?GGG?GCC?GAG?AAC?CTC?TTC?2164Gly?Ala?Asn?Val?Glu?Met?Ala?Glu?Glu?Ala?Gly?Ala?Glu?Ala?Leu?Phe695 700 705 710ATC?TTC?GGC?CTG?CGG?GTG?GAG?GAT?GTC?GAG?GCC?TTG?GAC?CGG?AAA?GGG?2212Ile?Phe?Gly?Leu?Arg?Val?Glu?Asp?Val?Glu?Ala?Leu?Asp?Arg?Lys?Gly

715 720 725TAC?AAT?GCC?AGG?GAG?TAC?TAC?GAC?CAC?CTG?CCC?GAG?CTG?AAG?CAG?GCC?2260Tyr?Asn?Ala?Arg?Glu?Tyr?Tyr?Asp?His?Leu?Pro?Glu?Leu?Lys?Gln?Ala

730 735 740GTG?GAC?CAG?ATC?AGC?AGT?GGC?TTT?TTT?TCT?CCC?AAG?GAG?CCA?GAC?TGC?2308Val?Asp?Gln?Ile?Ser?Ser?Gly?Phe?Phe?Ser?Pro?Lys?Glu?Pro?Asp?Cys

745 750 755TTC?AAG?GAC?ATC?GTG?AAC?ATG?CTG?ATG?CAC?CAT?GAC?AGG?TTC?AAG?GTG?2356Phe?Lys?Asp?Ile?Val?Asn?Met?Leu?Met?His?His?Asp?Arg?Phe?Lys?Val

760 765 770TTT?GCA?GAC?TAT?GAA?GCC?TAC?ATG?CAG?TGC?CAG?GCA?CAG?GTG?GAC?CAG?2404Phe?Ala?Asp?Tyr?Glu?Ala?Tyr?Met?Gln?Cys?Gln?Ala?Gln?Val?Asp?Gln775 780 785 790CTG?TAC?CGG?AAC?CCC?AAG?GAG?TGG?ACC?AAG?AAG?GTC?ATC?AGG?AAC?ATC?2452Leu?Tyr?Arg?Asn?Pro?Lys?Glu?Trp?Thr?Lys?Lys?Val?Ile?Arg?Asn?Ile

795 800 805GCC?TGC?TCG?GGC?AAG?TTC?TCC?AGT?GAC?CGG?ACC?ATC?ACG?GAG?TAT?GCA?2500Ala?Cys?Ser?Gly?Lys?Phe?Ser?Ser?Asp?Arg?Thr?Ile?Thr?Glu?Tyr?Ala

810 815 820CGG?GAG?ATC?TGG?GGT?GTG?GAG?CCC?TCC?GAC?CTG?CAG?ATC?CCG?CCC?CCC 2548Arg?Glu?Ile?Trp?Gly?Val?Glu?Pro?Ser?Asp?Leu?Gln?Ile?Pro?Pro?Pro

825 830 835AAC?ATC?CCC?CGG?GAC?TAGGCACACC?CTGCCTTGGC?GGGACCAGCG?GGCATTTGTT 2603Asn?Ile?Pro?Art?Asp

840TTCTTGCTGA CTTTGCACCT CCTTTTTTCC CCAAACACTT TGCCAGCCAC TGGTGGTCCC 2663TGCTTTTCTG AGTACCATGT TTCCAGGAGG GGCCATGGGG GTCAGGGTGG TTTTGAGAGA 2723GCAGGGTAAG GAAGGAATGT GCTAGAAGTG CTCCTAGTTT CTTGTAAAGG AAGCCAGAGT 2783TGACAGTACA AAGGGTCGTG GCCAGCCCTG CAGCTTCAGC ACCTGCCCCA CCCAGAGTGG 2843GAGTCAGGTG GAGCCACCTG CTGGGCTCCC CCAGAACTTT GCACACATCT TGCTATGTAT 2903TAGCCGATGT CTTTAGTGTT GAGCCTCTGG ATTCTGGGGT CTGGGCCAGT GGCCATAGTG 2963AAGCCTGGGA ATGAGTGTTA CTGCAGCATC TGGGCTGCCA GCCACAGGGA AGGGCCAAGC 3023CCCATGTAGC CCCAGTCATC CTGCCCAGCC CTGCCTCCTG GCCATGCCGG GAGGGGTCGG 3083ATCCTCTAGG CATCGCCTTC ACAGCCCCCT GCCCCCTGCC CTCTGTCCTG GCTCTGCACC 3143TGGTATATGG GTCATGGACC AGATGGGGCT TTCCCTTTGT AGCCATCCAA TGGGCATTGT 3203GTGGGTGCTT GGAACCCGGG ATGACTGAGG GGGACACTGG AGTGGGTGCT TGTGTCTGCT 3263GTCTCAGAGG CCTTGGTCAG GATGAAGTTG GCEGACACAG CTTAGCTTGG TTTTGCTTAT 3323TCAAAAGAGA AAATAACTAC ACATGGAAAT GAAACTAGCT GAAGCCTTTT CTTGTTTTAG 3383CAACTGAAAA TTGTACTTGG TCACTTTTGT GCTTGAGGAG GCCCATTTTC TGCCTGGCAG 3443GGGCAGGTCT GTGCCCTCCC GCTTGACTCC TGCTGTGTCC TGAGGTGCAT TTCCTGTTTG 3503TTACACACAA GCGCCAGGCT CCATTCTCCC TCCCTTTCCA CCAGTGCCAC AGCCTCGTCT 3563GGAAAAAGGA CCAGGGGTCC CGGAGGAACC CATTTGTGCT CTGCTTGGAC AGCAGGCCTG 3623GCACTGGGAG GTGGGGGTGA GCCCCTCACA GCCTTGCCCC TCCCCAAGGC TCGAACCTGC 3683CTCCCATTGC CCAAGAGAGA GGGCAGGGAA CAGGCTACTG TCCTTCCCTG TGGAATTGCC 3743GAGAAATCTA GCACCTTGCA TGCTGGATCT GGGCTGCGGG GAGGCTCTTT TTCTCCCTGG 3803CCTCCAGTGC CCACCAGGAG GATCTGCGCA CGGTGCACAG CCCACCAGAG CACTACAGCC 3863TTTTATTGAG TGGGGCAAGT GCTGGGCTGT GGTCGTGCCC TGACAGCATC TTCCCCAGGC 3923AGCGGCTCTG TGGAGGAGGC CATACTCCCC TAGTTGGCCA CTGGGGCCAC CACCCTGACC 3983ACCACTGTGC CCCTCATTGT TACTGCCTTG TGAGATAAAA ACTGATTAAA CCTTTGTGGC 4043TGTGGTTGGC TGAAAAAAAA AAA 406622

(ⅰ) sequence signature

(A) length: 843 amino acid

(B) type: amino acid

(C) topology: linearity

(ⅱ) molecule type: protein

(ⅲ) sequence description: No. 22 sequence Met Gly Glu Pro Leu Thr Asp Ser Glu Lys Arg Lys Gln Ile Ser Val1 5 10 15Arg Gly Leu Ala Gly Leu Gly Asp Val Ala Glu Val Arg Lys Ser Phe

20 25 30Asn?Arg?His?Leu?His?Phe?Thr?Leu?Val?Lys?Asp?Arg?Asn?Val?Ala?Thr

35 40 45Pro?Arg?Asp?Tyr?Phe?Phe?Ala?Leu?Ala?His?Thr?Val?Arg?Asp?His?Leu

50 55 60Val?Gly?Arg?Trp?Ile?Arg?Thr?Gln?Gln?His?Tyr?Tyr?Glu?Arg?Asp?Pro65 70 75 80Lys?Arg?Ile?Tyr?Tyr?Leu?Ser?Leu?Glu?Phe?Tyr?Met?Gly?Arg?Thr?Leu

85 90 95Gln?Asn?Thr?Met?Val?Asn?Leu?Gly?Leu?Gln?Asn?Ala?Cys?Asp?Glu?Ala

100 105 110Ile?Tyr?Gln?Leu?Gly?Leu?Asp?Leu?Glu?Glu?Leu?Glu?Glu?Ile?Glu?Glu

115 120 125Asp?Ala?Gly?Leu?Gly?Asn?Gly?Gly?Leu?Gly?Arg?Leu?Ala?Ala?Cys?Phe

130 135 140Leu?Asp?Ser?Met?Ala?Thr?Leu?Gly?Leu?Ala?Ala?Tyr?Gly?Tyr?Gly?Ile145 150 155 160Arg?Tyr?Glu?Phe?Gly?Ile?Phe?Asn?Gln?Lys?Ile?Val?Asn?Gly?Trp?Gln

165 170 175Val?Glu?Glu?Ala?Asp?Asp?Trp?Leu?Arg?Tyr?Gly?Asn?Pro?Trp?Glu?Lys

180 185 190Ala?Arg?Pro?Glu?Tyr?Met?Leu?Pro?Val?His?Phe?Tyr?Gly?Arg?Val?Glu

195 200 205His?Thr?Pro?Asp?Gly?Val?Lys?Trp?Leu?Asp?Thr?Gln?Val?Val?Leu?Ala

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

245 250 255Asp?Phe?Asn?Val?Gly?Asp?Tyr?Ile?Glu?Ala?Val?Leu?Asp?Arg?Asn?Leu

260 265 270Ala?Glu?Asn?Ile?Ser?Arg?Val?Leu?Tyr?Pro?Asn?Asp?Asn?Phe?Phe?Glu

275 280 285Gly?Lys?Glu?Leu?Arg?Leu?Lys?Gln?Glu?Tyr?Phe?Val?Val?Ala?Ala?Thr

290 295 300Leu?Gln?Asp?Ile?Ile?Arg?Arg?Phe?Lys?Ser?Ser?Lys?Phe?Gly?Cys?Arg305 310 315 320Asp?Pro?Val?Arg?Thr?Cys?Phe?Glu?Thr?Phe?Pro?Asp?Lys?Val?Ala?Ile

325 330 335Gln?Leu?Asn?Asp?Thr?His?Pro?Ala?Leu?Ser?Ile?Pro?Glu?Leu?Met?Arg

340 345 350Ile?Leu?Val?Asp?Val?Glu?Lys?Val?Asp?Trp?Asp?Lys?Ala?Trp?Glu?Ile

355 360 365Thr?Lys?Lys?Thr?Cys?Ala?Tyr?Thr?Asn?His?Thr?Val?Leu?Pro?Glu?Ala

370 375 380Leu?Glu?Arg?Trp?Pro?Val?Ser?Met?Phe?Glu?Lys?Leu?Leu?Pro?Arg?His385 390 395 400Leu?Glu?Ile?Ile?Tyr?Ala?Ile?Asn?Gln?Arg?His?Leu?Asp?His?Val?Ala

405 410 415Ala?Leu?Phe?Pro?Gly?Asp?Val?Asp?Arg?Leu?Arg?Arg?Met?Ser?Val?Ile

420 425 430Glu?Glu?Gly?Asp?Cys?Lys?Arg?Ile?Asn?Met?Ala?His?Leu?Cys?Val?Ile

435 440 445Gly?Ser?His?Ala?Val?Asn?Gly?Val?Ala?Arg?Ile?His?Ser?Glu?Ile?Val

450 455 460Lys?Gln?Ser?Val?Phe?Lys?Asp?Phe?Tyr?Glu?Leu?Glu?Pro?Glu?Lys?Phe465 470 475 480Gln?Asn?Lys?Thr?Asn?Gly?Ile?Thr?Pro?Arg?Arg?Trp?Leu?Leu?Leu?Cys

485 490 495Asn?Pro?Gly?Leu?Ala?Asp?Thr?Ile?Val?Glu?Lys?Ile?Gly?Glu?Glu?Phe

500 505 510Leu?Thr?Asp?Leu?Ser?Gln?Leu?Lys?Lys?Leu?Leu?Pro?Leu?Val?Ser?Asp

515 520 525Glu?Val?Phe?Ile?Arg?Asp?Val?Ala?Lys?Val?Lys?Gln?Glu?Asn?Lys?Leu

530 535 540Lys?Phe?Ser?Ala?Phe?Leu?Glu?Lys?Glu?Tyr?Lys?Val?Lys?Ile?Asn?Pro545 550 555 560Ser?Ser?Met?Phe?Asp?Val?His?Val?Lys?Arg?Lle?His?Glu?Tyr?Lys?Arg

565 570 575Gln?Leu?Leu?Asn?Cys?Leu?His?Val?Val?Thr?Leu?Tyr?Asn?Arg?Ile?Lys

580 585 590Arg?Asp?Pro?Ala?Lys?Ala?Phe?Val?Pro?Arg?Thr?Val?Met?Ile?Gly?Gly

595 600 605Lys?Ala?Ala?Pro?Gly?Tyr?His?Met?Ala?Lys?Leu?Ile?Ile?Lys?Leu?Val

610 615 620Thr?Ser?Ile?Gly?Asp?Val?Val?Asn?His?Asp?Pro?Val?Val?Gly?Asp?Arg625 630 635 640Leu?Lys?Val?Ile?Phe?Leu?Glu?Asn?Tyr?Arg?Val?Ser?Leu?Ala?Glu?Lys

645 650 655Val?Ile?Pro?Ala?Ala?Asp?Leu?Ser?Gln?Gln?Ile?Ser?Thr?Ala?Gly?Thr

660 665 670Glu?Ala?Ser?Gly?Thr?Gly?Asn?Met?Lys?Phe?Met?Leu?Asn?Gly?Ala?Leu

675 680 685Thr?Ile?Gly?Thr?Met?Asp?Gly?Ala?Asn?Val?Glu?Met?Ala?Glu?Glu?Ala

690 695 700Gly?Ala?Glu?Asn?Leu?Phe?Ile?Phe?Gly?Leu?Arg?Val?Glu?Asp?Val?Glu705 710 715 720Ala?Leu?Asp?Arg?Lys?Gly?Tyr?Asn?Ala?Arg?Glu?Tyr?Tyr?Asp?His?Leu

725 730 735Pro?Glu?Leu?Lys?Gln?Ala?Val?Asp?Gln?Ile?Ser?Ser?Gly?Phe?Phe?Ser

740 745 750Pro?Lys?Glu?Pro?Asp?Cys?Phe?Lys?Asp?Ile?Val?Asn?Met?Leu?Met?His

755 760 765His?Asp?Arg?Phe?Lys?Val?Phe?Ala?Asp?Tyr?Glu?Ala?Tyr?Met?Gln?Cys

770 775 780Gln?Ala?Gln?Val?Asp?Gln?Leu?Tyr?Arg?Asn?Pro?Lys?Glu?Trp?Thr?Lys785 790 795 800Lys?Val?Ile?Arg?Asn?Ile?Ala?Cys?Ser?Gly?Lys?Phe?Ser?Ser?Asp?Arg

805 810 815Thr?Ile?Thr?Glu?Tyr?Ala?Arg?Glu?Ile?Trp?Gly?Val?Glu?Pro?Ser?Asp

820 825 830Leu?Gln?Ile?Pro?Pro?Pro?Asn?Ile?Pro?Arg?Asp

The information of 835 840 No. 23 sequences

(ⅰ) sequence signature

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topology: linearity

(ⅱ) molecule type: cDNA

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: the information of No. 24 sequence of No. 23 sequence GGGCTAGGCG ACGTGGCCGA GGTG 24

(ⅰ) sequence signature

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topology: linearity

(ⅱ) molecule type: cDNA

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: the information of No. 25 sequence of No. 24 sequence GCGGGGCTAG GCGACGTGGC CGAGGTG 27

(ⅰ) sequence signature

(A) length: 30 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topology: linearity

(ⅱ) molecule type: cDNA

(ⅲ) hypothesis: do not have

(ⅳ) antisense: do not have

(ⅴ) clip types: inside

(ⅵ) source:

(A) structure: Homo sapiens

(ⅶ) sequence description: No. 25 sequence C TGGCGGGGC TAGGCGACGT GGCCGAGGTG 30

Claims (40)

1. the fragment of a brain glycogen phosphorylase that is separated, this fragment comprises the molecule with No. 15 sequence, wherein said fragment comprises and is no more than 75% No. 21 sequence.

2. the fragment that is separated according to claim 1, wherein said fragment comprise 100 amino acid that are no more than No. 21 sequence.

3. the fragment that is separated according to claim 1, wherein said fragment comprise 7-100 amino acid whose molecule, and No. 15 sequence.

4. the fragment that is separated according to claim 3, wherein said fragment comprise having the molecule that is selected from the sequence in a group that is made up of No. 15, No. 14, No. 13, No. 12 and No. 5 sequence.

5. the fragment that is separated according to claim 4, wherein said fragment comprise having the molecule that is selected from the sequence in a group that is made up of No. 14, No. 13 and No. 12 sequence.

6. nucleic acid encoding that is separated, wherein said polypeptide is selected from by in the group that any one described fragment is formed among the claim 1-5.

7. the nucleic acid that is separated according to claim 6, wherein said nucleic acid encoding are selected from the polypeptide in the group of being made up of any fragment of claim 4 or 5.

8. the nucleic acid that is separated according to claim 6, wherein said nucleic acid comprise that coding has the molecule that is selected from by the polypeptide of sequence in the group that No. 14, No. 13 and No. 12 sequence is formed.

9. expression vector, but comprise with the promotor workability the nucleic acid that is separated of the claim 6 that couples together.

10. expression vector according to claim 9, the wherein said nucleic acid that is separated comprise No. 23, No. 24 or No. 25 sequence.

11. according to Claim 8,9 or 10 described expression vectors, wherein said carrier further comprises the nucleic acid of the HLA-A2 that encodes.

12. expression vector institute's transfection or transformed host cells in the group that the expression vector of the expression vector of a selected free claim 9, the expression vector of claim 10 and claim 11 is formed.

13. one kind optionally strengthens in the T cell colony the brain glycogen phosphorylase Tumor rejection antigen is the method for the molten born of the same parents' property T of specific cell cell, this method comprises:

Allow the T cell colony that is separated provide the preparation of the complex body of molecule formation to contact with brain glycogen phosphorylase Tumor rejection antigen and HLA are provided, the consumption of described preparation is the dosage that is enough to optionally strengthen the molten born of the same parents' property T of described cell cell in the described T cell colony that is separated.

14. method according to claim 13, it is HLA-A2 that wherein said HLA provides molecule, and wherein said brain glycogen phosphorylase Tumor rejection antigen is the peptide that comprises No. 15 sequence.

15. comprising, method according to claim 14, wherein said peptide have the molecule that is selected from by the sequence in the group that No. 15, No. 14, No. 13, No. 12 and No. 5 sequence is formed.

16. comprising, method according to claim 15, wherein said peptide have the molecule that is selected from by the sequence in the group that No. 14, No. 13 and No. 12 sequence is formed.

17. a diagnosis is the method for the disease of feature to express the brain glycogen phosphorylase Tumor rejection antigen, this method comprises:

With pick up from patient's biological sample be that specific preparation contacts to the brain glycogen phosphorylase Tumor rejection antigen, wherein said biological sample is separated from non-brain, non-retinal pigment epithelium tissue, and

Determine that the interaction between said preparation and the brain glycogen phosphorylase Tumor rejection antigen comes disease is diagnosed.

18. method according to claim 17, wherein said brain glycogen phosphorylase Tumor rejection antigen are the amino acid whose peptides that comprises No. 15 sequence.

19. method according to claim 18, wherein said peptide are the amino acid between 7-100 of No. 21 sequence.

20. comprising, method according to claim 19, wherein said peptide have the molecule that is selected from by the sequence in the group that No. 15, No. 14, No. 13, No. 12 and No. 5 sequence is formed.

21. comprising, method according to claim 20, wherein said peptide have the molecule that is selected from by the sequence in the group that No. 14, No. 13 and No. 12 sequence is formed.

22. a diagnosis is the method for the disease of feature with expression with the brain glycogen phosphorylase Tumor rejection antigen that the HLA-A2 molecule forms complex body, this method comprises:

With pick up from patient's biological sample with contact with complex body bonded preparation; And

Determine combining disease is diagnosed between complex body and the preparation.

23. method according to claim 22, wherein said brain glycogen phosphorylase Tumor rejection antigen comprises the amino acid of No. 15 sequence.

24. method according to claim 23, wherein said peptide comprise the successive amino acid between 7-100 of No. 21 sequence.

25. comprising, method according to claim 24, wherein said peptide have the molecule that is selected from by the sequence in the group that No. 15, No. 14, No. 13, No. 12 and No. 5 sequence is formed.

26. comprising, method according to claim 25, wherein said peptide have the molecule that is selected from by the sequence in the group that No. 14, No. 13 and No. 12 sequence is formed.

27. the application of preparation in the preparation medicine, described preparation comprises the nucleic acid molecule that is separated of coding brain glycogen phosphorylase Tumor rejection antigen, or comprised the peptide of brain glycogen phosphorylase Tumor rejection antigen, and they all optionally strengthen patient's the HLA and the complex body of brain glycogen phosphorylase Tumor rejection antigen.

28. application according to claim 27, wherein said brain glycogen phosphorylase Tumor rejection antigen are the amino acid whose peptides that comprises No. 15 sequence.

29. comprising, application according to claim 28, wherein said peptide have the molecule that is selected from by the sequence in the group that No. 15, No. 14, No. 13, No. 12 and No. 5 sequence is formed.

30. comprising, application according to claim 29, wherein said peptide have the molecule that is selected from by the sequence in the group that No. 14, No. 13 and No. 12 sequence is formed.

31. the complex body to HLA and brain glycogen phosphorylase Tumor rejection antigen is specific from the application of the molten born of the same parents' property T of body cell in the preparation medicine.

32. application according to claim 31, it is HLA-A2 that wherein said HLA provides molecule, and wherein said brain glycogen phosphorylase Tumor rejection antigen is the peptide that comprises the amino acid whose molecule with No. 15 sequence.

33. application according to claim 32, wherein said peptide are the successive amino acid between 7-100 of No. 21 sequence.

34. comprising, application according to claim 33, wherein said peptide have the molecule that is selected from by the sequence in the group that No. 15, No. 14, No. 13, No. 12 and No. 5 sequence is formed.

35. comprising, application according to claim 34, wherein said peptide have the molecule that is selected from by the sequence in the group that No. 14, No. 13 and No. 12 sequence is formed.

36. a diagnosis is the method for the disease of feature to express brain glycogen phosphorylase, this method comprises:

With pick up from patient's biological sample with contact with brain glycogen phosphorylase bonded preparation, wherein said biological sample is separated from non-cerebral tissue, non-retinal pigment epithelium tissue, non-renal cell carcinoma tissue, non-liver cancer tissue, non-stomach organization; And

Determine combining disease is diagnosed between brain glycogen phosphorylase and the described preparation.

37. one kind is the diagnostic method of the disease of feature with the nucleic acid of expressing the coding brain glycogen phosphorylase, this method comprises:

With pick up from patient's biological sample be that specific preparation contacts to described nucleic acid or its expression product, wherein said biological sample is separated from non-cerebral tissue, non-retinal epithelium tissue, non-renal cell carcinoma tissue, non-liver cancer tissue, non-stomach organization, and wherein said nucleic acid under stringent condition with comprise the molecular hybridization of the described nucleotide sequence of claim 8; And

Determine that the interaction of described preparation between described nucleic acid or its expression product comes disease is diagnosed.

38. a medicinal compositions, comprise the pharmacology effective dose with HLA molecule bonded brain glycogen phosphorylase or its fragment, and pharmacology acceptable carrier.

39., wherein saidly comprise the amino acid whose peptide that contains No. 15 sequence with HLA molecule bonded brain glycogen phosphorylase or its fragment according to the described medicinal compositions of claim 38.

40. a medicinal compositions, the complex body to HLA molecule and brain glycogen phosphorylase Tumor rejection antigen that comprises the pharmacology effective dose are specific from body T cell, and the pharmacology acceptable carrier.

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