CN1326875C - Tumor antigen protein and tumor antigen peptide - Google Patents
Tumor antigen protein and tumor antigen peptide Download PDFInfo
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- CN1326875C CN1326875C CNB03136411XA CN03136411A CN1326875C CN 1326875 C CN1326875 C CN 1326875C CN B03136411X A CNB03136411X A CN B03136411XA CN 03136411 A CN03136411 A CN 03136411A CN 1326875 C CN1326875 C CN 1326875C
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Abstract
The present invention relates to tumor antigen protein and tumor antigen peptide. The tumor antigen protein can generate a peptide fragment which is combined with molecules in the major histocompatibility complex (MHC)-I class and is identified by a T cell in a combination state, a DNA sequence for encoding the tumor antigen protein has a nucleotide sequence of a sequence with the accession number in a gene library of FATEAF249872, and the tumor antigen protein / the tumor antigen peptide can be used for preparing medicine for treating liver cancer or intestinal cancer.
Description
Technical field
The present invention relates to a kind of tumor antigen protein matter and tumor antigen peptide, particularly relate to a kind of tumor antigen protein matter and tumor antigen peptide that is used for the treatment of liver cancer and intestinal cancer.
Technical background
Malignant tumour is a big class disease that threatens human health.Tumour be a very complicated process.The important factor that tumour cell can take place and make progress is that it can escape the supervision effect of body immune system, therefore how to excite and the tumour cell that arouses the immune system recognition of body and repel " non-oneself " composition is a breach seeking new tumor therapeuticing method.At the initial stage that the immunotherapy of tumour is studied, people have been developed a lot of ways and have been come the immunity system of excitating organism to repel the tumour cell of " non-own " composition, although these methods have obtained better curative effect in the treatment of animal tumor model, in the treatment of human tumor, still lack significant effect.
The intravital immunity system of known person comprises humoral immunization and cellular immunization, and especially T cellular immunization suppresses to play an important role to killing and wounding of tumour.Along with the understanding of people to immune response essence, it is found that no matter how complicated the process of immunne response is, finally cause the T cell activation and produce being exactly to combine and forming antigenic peptide complexes and costimulatory signal of lethal effect with major histocompatibility complex MHC, that is: cytotoxic T cell (CTL) is by the TXi Baoshouti (TCR) on self surface, the complex body that recognition expression combines with major histocompatibility complex (MHC) I class antigen and forms in the tumor antigen protein matter/peptide of tumor cell surface, thus killing tumor cell attacked.
The generation of tumor antigen protein matter/peptide is to be expressed as tumour antigen earlier by tumour cell, it is resolved into the antigen peptide of 8～10 amino acid sizes by various proteolytic enzyme in cell, these antigen peptide produce the MHCI quasi-molecule with the while and combine in endoplasmic reticulum, be expressed in the surface of cell by Golgi complex, finally offer to the special CTL of tumour antigen, thereby bring out immunne response, kill and wound the removing tumour cell.The proteantigen molecule that thisly be present in the tumour cell, can combine and bring out organism immune response with the MHC molecule is tumour antigen.Can only produce a immune response with these antigen induction bodies at tumour cell, thus the intravital tumour cell of specific killing and reach the effect that treatment or control tumour cell shift.Therefore in recent decades, people are seeking on tomour specific and/or the dependency antigen with focusing on of immunization method treatment tumour.
In nineteen fifties, people such as Foley self the tumour cell immunity of the mouse that produces tumour through chemomorphosis with deactivation after, the tumour cell of living for this mouse inoculation again, these tumour cells are ostracised and can not be grown into tumour, mouse then survives; Contrast with it, behind the tumour cell that self tumour cell mice immunized inoculation of deactivation useless is lived, tumour cell is not ostracised and is grown into tumour, and mouse is then dead.This experiment has proved that with compellent evidence there is tumour specific antigen in the tumour of chemomorphosis; but this protective reaction has accurate specificity; promptly only at the tumour of same type and not at different types of tumors, even to also not having provide protection with same carcinogen dissimilar tumours of inductive on same mouse.
1991, T.Boon etc. identified tumor antigen protein matter first from human melanoma cell, and called after MAGE (science, 254:1643-1647,1991).Subsequently, people identify out some other tumor antigen protein matter again, can be divided into the tumor antigen protein matter of tumor-testis (CT) antigen protein, differentiation antigen protein, sudden change, tumor antigen protein matter and virus antigen protein five classes of expressing excessively.Wherein, being applied to is tumor-testis (CT) antigen protein clinically the most widely.
Tumor-testis (CT) antigen protein is a maximum class in the tumour antigen of identifying at present, the characteristics of their encoding genes are in the tumour of a lot of types expression to be arranged all, as expression is all arranged in melanoma, lung cancer, sarcoma and bladder cancer, but do not express in the healthy tissues except that testis, a certain amount of expression is arranged in placenta, ovary, pancreas.Because testis is immunity special permission position, so this class antigen is considered to the antigen of tumour-specific in treatment, is a class antigen that is hopeful to be used as the immunotherapy of tumors purposes most.Try out at present in clinically also mainly be exactly this class antigen, as MAGE-1 and NY-ESO-1 promptly is the expression that high positive rate is arranged in the tumour of certain type, the antigen protein that good immunogenicity is arranged again has good prospect when being used for the immunotherapy of tumour.
Up to the present, the most representative method of tumor antigen protein matter of identifying is: based on the library screening method on the specificity cellular immunity response basis, based on cDNA expression library sieve method, combined peptide library screening, sour elution method and bioinformatics method on the humoral immunoresponse(HI) basis.
Liver cancer and intestinal cancer are the most common and virulent tumours the most in the human tumor, and especially in the last few years, sickness rate increased gradually.Liver cancer is because the grade of malignancy height, growth rapidly, in case find to be middle and advanced stage just, at present, to the treatment of liver cancer mainly is local therapeutic approaches such as traditional excision and radiotherapy, but all entered middle and advanced stage when making a definite diagnosis, and liver cancer there is suitable resistivity to radiotherapy and chemotherapy owing to most of patient.Therefore the effect and the prognosis of treatment are often relatively poor, and it is very low that a year of hepatocarcinoma patient is compared with the survival rate of other all kinds of tumour patients with five-year survival rate.Breakthrough or the new methods of treatment sought on the methods of treatment are the targets that the clinical tumor scholar pursues always, people's early diagnosis and good prognosis of patient that new method improves liver cancer that wait in expectation, and postoperative is removed tumour cell residual in the body and is prevented the transfer of tumour cell.
Summary of the invention
The object of the present invention is to provide a kind of tumor antigen protein matter and tumor antigen peptide that can be used for liver cancer and intestinal cancer treatment.
The objective of the invention is to realize by the following technical solutions:
The invention provides a kind of tumor antigen protein matter, this protein is by decomposing in the cell, can produce the peptide fragment combine with major histocompatibility complex (MHC)-I quasi-molecule, can be discerned by the T cell at bonding state, it has following (a) or (b) described aminoacid sequence:
A) has the aminoacid sequence shown in the sequence 1;
B) with the aminoacid sequence in (a) through the replacement of one or several amino-acid residue, disappearance or add the derived protein that sudden change is produced, and this derived protein has identical functions with (a) protein.
The invention provides a kind of dna sequence dna, its described tumor antigen protein matter of encoding.
Dna sequence dna of the present invention has the nucleotide sequence shown in the sequence 2, and its accession number in gene library is: FATE AF249872.
The invention provides of the application of described protein as tumor-testis antigen.
The invention provides of the application of described gene as the tumor-testis antigen gene.
The invention provides the tumor antigen peptide of the peptide segment same function that a kind of and described tumor antigen protein matter produced, it has the 140th～the 148th aminoacid sequence of aminoacid sequence in the sequence 1.
The application of tumor antigen protein matter of the present invention in preparation treatment liver-cancer medicine.
The application of tumor antigen protein matter of the present invention in preparation treatment bowelcancer medicine.
The application of tumor antigen peptide of the present invention in preparation treatment liver-cancer medicine.
The application of tumor antigen peptide of the present invention in preparation treatment liver-cancer medicine.
The advantage of tumor antigen protein matter provided by the invention and tumor antigen peptide is: the present invention is the tumor-testis antigen gene with the FATE gene identification first; When using immunization method treatment cancer, can produce a immune response with these antigen induction bodies at tumour cell, thereby do not killing and wounding the Normocellular while the intravital tumour cell of specific killing and reach the effect that treatment or control tumour cell shift; Simultaneously, can be by to this tumor antigen gene or detection of antibodies, to generation, the development of tumour and whether have to shift and diagnose.
Description of drawings
Fig. 1 is the RT-PCR detected result of the gene FATE of code book invention antigen protein 16 kinds of adult's healthy tissuess; Among the figure 1～16 represents adult's healthy tissues spleen, prostate gland, ovary, small intestine, large intestine, white corpuscle, testis, heart, lung, liver, brain, kidney, placenta, skeletal muscle, pancreas, thymus gland respectively;
Fig. 2 is that the gene FATE of code book invention antigen protein is the cancerous tissue of liver cancer and the RT-PCR result of paired cancer beside organism; Ca among the figure represents cancerous tissue, and adj represents paired cancer beside organism;
Fig. 3 is the Northern blot result of the gene FATE of code book invention antigen protein in liver cancer tissue and paired cancer beside organism; Ca among the figure represents cancerous tissue, and adj represents paired cancer beside organism;
Fig. 4 is the recombinant protein of antigen protein of the present invention in intestinal bacteria and the representative result of hepatocarcinoma patient sero-reaction that detects by Western blot method; M among the figure represents the protein molecular weight standard reference, intestinal bacteria lysate that the tumor-testis antigen recombinant protein among the present invention is arranged and the result who resists histidine-tagged monoclonal antibody reactive are expressed in 1 representative, 2 represent the result of tumor-testis antigen recombinant protein and anti-histidine-tagged monoclonal antibody reactive among the present invention of purifying, 3 representatives are expressed the intestinal bacteria lysate of the tumor-testis antigen recombinant protein among the present invention and the result of hepatocarcinoma patient sero-reaction, 4 represent the tumor-testis antigen recombinant protein among the present invention of purifying and the result of hepatocarcinoma patient sero-reaction, on behalf of transfection, 5 the intestinal bacteria lysate of empty expression vector and the result of hepatocarcinoma patient sero-reaction are arranged, and on behalf of transfection, 6 the intestinal bacteria lysate of irrelevant albumen contrast expression vector and the result of hepatocarcinoma patient sero-reaction are arranged;
Fig. 5 is by the recombinant protein of tumor-testis antigen protein in intestinal bacteria and the result of hepatocarcinoma patient sero-reaction among the present invention of ELISA method detection; Wherein X-axis is represented the extent of dilution of serum, the absorbancy when on behalf of wavelength, Y-axis be 410nm; "-◆-" " ■-" " ▲-" represent the result of recombinant protein and 3 different tumour patient sero-reactions respectively.
Embodiment
The screening of embodiment 1, FATE gene
Be expressed as descriptor with testes specificity and in the gene database (GenBank) of American National biotechnology information center website (http://www.ncbi.nlm.nih.gov) and literature search database (PubMed), retrieve the gene of testis specific expression in people's healthy tissues that those have had strict difinition, but it must be up to the present without any the report relevant with tumour.We have obtained to comprise the gene of several testis specific expressions in people's healthy tissues of FATE gene thus, their target gene as our next step Unigene and SAGE database analysis.
Unigene is a network tool that all Genbank sequences that will derive from same gene are sorted out automatically.In the Unigene database, each Unigene gene include derive from various tissues but belong to the sequence that the segment of same gene varies in size, the tissue-derived and expressing information of these sequences is all indicated.SAGE (serial analysis of genetic expression) database is one can carry out the express spectra of a certain specific gene quantitatively and online tool qualitatively.Therefore, application Unigene and SAGE database just can be analyzed the time and space expression of a certain special genes, thereby obtain its correlation function information.In process of the present invention, three standards are used to screen candidate gene, obtain tumor-testis (CT) antigen gene of purpose with expectation: 1. candidate gene must be the gene of testis specific expression in people's healthy tissues of strict difinition, but it must be up to the present without any the report relevant with tumour.2. in the Unigene database analysis in process of the present invention, must contain the expressed sequence tag (EST) 3. in the SAGE database analysis in process of the present invention that derives from tumour in the Unigene data of candidate gene, if there is the SAGE label in candidate gene, have only the SAGE label of strict corresponding candidate gene just can be applied in the analysis of candidate gene.After above-mentioned three standards were used, candidate gene FATE was considered to most possible tumor-testis antigen gene.In the Unigene of FATE gene data, have 33 EST fragments, though 31 EST wherein derive from testis tissue, other 2 is to derive from tumor tissues; In the SAGE of FATE gene data, the FATE gene does not derive from the SAGE label of tumor tissues.Based on above-mentioned analysis, think that the FATE gene might be the goal gene one tumor-testis antigen gene that desire is sought among the present invention, next step is proved contrived experiment.
CDNA (the spleen of 16 kinds of commercial people's healthy tissuess of the CLONTECH of biotech company, prostate gland, testis, ovary, small intestine, large intestine, white corpuscle, heart, lung, liver, brain, kidney, pancreas, placenta, skeletal muscle, thymus gland), derive from the cancerous tissue of 62 routine hepatocarcinoma patients and the cDNA of paired cancer beside organism, derive from the cancerous tissue of 14 routine lung cancer patients and the cDNA of paired cancer beside organism, the cDNA that derives from the cancerous tissue of 14 routine Patients with Gastric Cancer and paired cancer beside organism with derive from 14 routine intestinal cancer patients' cancerous tissue and the cDNA of paired cancer beside organism is used to detect the expression of FATE gene at healthy tissues and tumor tissues.
Carry out the condition of RT-PCR among the present invention: use the advanced warm start archaeal dna polymerase of CLONTECH company, 94 ℃, 15 seconds; 68 ℃, 30 seconds; 72 ℃, 30 seconds, 30 circulations, afterwards, 72 ℃, 6 minutes.Carry out the primer sequence of RT-PCR, forward primer: ctg ttc ctg gca ccc tgt gca tcc; Reverse primer: gat gcc gcc atgctg ttc acc c.Found that: the FATE gene is expressed in 66% liver cancer sample, 21% intestinal cancer, and (as Fig. 2) do not express in paired cancer beside organism with it; In 16 kinds of important healthy tissuess of adult, find among the present invention that the FATE gene also has expression in pancreas, its expression amount approximately is 1/5 (as Fig. 1) of testis tissue expression amount, and the expression amount of FATE gene in liver cancer is consistent with the expression amount at testis, and hangs down about one times than testis in intestinal cancer.Based on above experimental data, proof is consistent with Unigene and SAGE database analysis result among the embodiment 1 in the present embodiment, and the FATE gene is a new tumor-testis antigen gene.
From liver cancer and intestinal cancer patient's cancerous tissue and paired cancer beside organism, extract total RNA with TRIZOL reagent (PROMEGA), then use mRNA separating kit (PROMEGA) separating mRNA from total RNA.Under the condition that methane amide, formaldehyde exist,, carry out forwarding on the Hybond-N+ nylon membrane (Amersham) after the agarose electrophoresis, fix with UV-crosslinked method with the mRNA sex change of 2ug.With the dna fragmentation of coming out that increases among digoxin dna probe labelling kit (Roche) the mark embodiment 2, to make dna probe, hybridize according to the Northern hybridizing method of routine and the mRNA on the film, carry out radioautograph afterwards, detect the mRNA of the Testiculo-tumor antigen protein plasmagene FATE among the present invention.Then, in 1%SDS solution, boil detecting the used film of this gene mRNA, remove the probe of digoxigenin labeled after, make probe with the house-keeping gene G3PDH of mark, use the same method and carry out Northern hybridization, detected mRNA makes positive control.The result as shown in Figure 3.From this result as can be seen, the mRNA of tumor antigen protein plasmagene of the present invention only expresses in cancerous tissue, and paired cancer beside organism does not express.Consistent with the characteristic of tumor-testis antigen gene.
Use pQE30 prokaryotic expression carrier (INVOTRIGEN), in coli strain M15 (INVOTRIGEN), under 37 ℃ of growth conditionss, IPTG induced recombinant protein expression 6 hours.The SDS-PAGE protein electrophoresis is identified Recombinant Protein Expression.Afterwards, application of nickel ion affinity chromatography pillar (INVOTRIGEN) with recombinant protein from the intestinal bacteria whole protein, separate, purifying comes out.The monoclonal antibody of 6 Histidines that have with anti-recombinant protein by the Western hybridizing method, is identified recombinant protein.The plasmid pQE30-FATE of expressing tumor-testis antigen FATE and the coli strain M15 that contains plasmid pQE30-FATE are stored in-80 ℃ of cryogenic refrigerators that the immunity of Department Of Medicine, Peking University's preclinical medicine institute of the People's Republic of China (PRC) is T cell research chamber, and date saved is on May 26th, 2002.
With reference to The Journal of Experimental Medicine, 187:1349, the method in 1998, the serum of screening FATE mRNA transcript male tumour patient finds to exist the antibody (Fig. 4,5) of anti-FATE recombinant protein in the serum of tumour patient.Therefore, FATE is a new tumor-testis antigen, has the induced tumor patient and produces immunne response, repels the ability of killing tumor cell, and the potential of the clinical immunotherapy of the tumour of being applied to is arranged.
Formation and the CTL killing experiments in vitro of embodiment 7, the external evoked CTL of application tumor antigen peptide
With reference to the experimental technique in the Chinese general surgery magazine (17:218,2002).PBMC (peripheral blood lymphocytes) 2ml of 2 * 106/ml concentration is incubated in 24 well culture plates, and the action effect lymphocyte simultaneously, presses 2 * 10 with other a part of PBMC
6/ ml concentration dilution adds the tumor antigen peptide 40ug/ml with aminoacid sequence of the 140th～the 148th in the sequence 1 in RPMI 1640 substratum, 37 ℃ hatch 2 hours after, through 30GY
60The Co gammairradiation, centrifugal, flush away residue peptide, be used as antigen deduction cell, it by effector cell: APC 13: 1 APC cell count, the antigen cell of deducting a percentage is added above-mentioned effector cell's co-cultivation, after 24 hours, add IL-2 (Australian Ludwig Institute for Cancer Research provides) 25U/ml and continue to cultivate.Later on every 7 days, by the effector cell: antigen deduction cell was that 1.3: 1 antigen deduction cell quantity adds the antigen cell of deducting a percentage among the effector cell and stimulates 3 times again, detected the lethal effect of CTL in the 28th day.Killing experiments is undertaken by following: (1) adopts the on-radiation cell toxicant to analyze box, measures CTL and target cell and cultivates the lethal effect that back LDH releasing value detects CTL altogether.Operate according to the test kit recommended program.Used substratum is the no phenol red RPMI 1640 that contains 3%FCS, and 3 multiple holes are all established in every kind of reaction, measures the spontaneous releasing value of target cell, the maximum releasing value of target cell, the spontaneous releasing value of effector cell, volume correction value and substratum background value simultaneously.Use following formula and calculate lethal effect: lethal effect (%)=(the spontaneous releasing value of the spontaneous releasing value-target cell of experimental value-effector cell)/(the spontaneous releasing value of the maximum releasing value-target cell of target cell) * 100.(2) after mixed lymphocytes and target cell are cultivated 4 hours altogether, observe the morphological change of lymphocyte and target cell.After 28 days, peripheral blood lymphocytes has increased 32 times, the ratio of CD3 positive cell 16% (from 54% to 70%) that risen wherein, CTL ratio 20% (from 3% to 56%) that risen.As the effector cell: when target cell is 10: 1, CTL to tumor antigen peptide (biochemical company limited is synthetic by the Shanghai gill) hatch be 62.5% from the lymphoblastic lethal effect of body, lethal effect to tumour antigen FATE male tumour cell is 40.2%, and both are all apparently higher than to the lethal effect (1.6%) from the tumour cell of lymphoblastic lethal effect of body (17.9%) and tumour antigen FATE feminine gender; As the effector cell: when target cell is 3.3: 1, CTL to tumor antigen peptide hatch be 53.6% from the lymphoblastic lethal effect of body, apparently higher than to from the lymphoblastic lethal effect of body (15.6%).The tumour antigen Toplink of these presentation of results tumour antigens FATE effectively induces the CTL with specific killing tumour cell from the PBMC of tumour patient, thereby strengthens the ability that tumour patient is removed tumour.
With reference to the experimental technique in the Chinese Medical Journal (81:1234,2001).Carry out the quantitative of IFN-γ with enzyme linked immunological spot detection method (ELISPOT).The flat nitrocellulose plate in 96 holes (available from French Millipore company), (be dissolved in the pH9.6 carbonate buffer solution with anti-people IFN γ monoclonal antibody, final concentration is 2ug/ml) the bag quilt, 4 ℃ are spent the night: after phosphate buffered saline buffer (PBS) washing through containing 0.05%Tween 20 in the 2nd day, with the RPMI1640 nutrient solution room temperature lucifuge sealing that contains 10%AB serum 1 hour, with 0.05%Tween PBS washing, add again through 7 days inductive effector cells (effector cells, E) 5 * 10
4Individual/hole.After this experiment divides two groups to be carried out, and promptly effector cell and T2 groups of cells (E+T2), effector cell and T2 cell load tumor antigen peptide group (E+T2 tumor antigen peptide), does two multiple holes for every group.Simple T2 cell and the T2 cell that loaded the 10ug/ml tumor antigen peptide in serum-free RPMI 1640,37 ℃, 5%CO
2Hatched warp under the condition 2 hours
60Co gamma-rays 100GY irradiation, with after the unnecessary antigen peptide of serum-free RPMI 1640 flush awaies as irritation cell, with 1 * 10
5Individual/hole adds each effector cell hole; This reaction system in the RPMI RPMI-1640 of not factor-containing and serum, 37 ℃, 5%CO
2After hatching 18～20h under the condition, fully wash culture plate to remove residual cell with 0.05%Tween PBS: it is anti-to add the biotin labeled anti-people IFN γ of 0.2ug/ml two subsequently, hatches 2 hours in 37 ℃, washes plate; The Streptavidin 1ug/m that adds alkali phosphatase enzyme mark again, lucifuge was hatched 1 hour under the room temperature; Wash plate, add substrate colour developing liquid, lucifuge colour developing 5 minutes is with tap water flushing, termination reaction.After the question response plate dries, count every hole spot number of purple darkly down, obtain the mean value in two multiple holes in dissecting microscope.E+T2 tumor antigen peptide group spot number deducts E+T2 group spot number and is the special CTL cell frequency of tumour antigen.During ELISPOT detects, to produce the CD8 of IFN γ
+The frequency of T cell is an index, measures the respondent to tumour antigen FATE respectively in the patient of expression of tumor tissue FATE mRNA, does not all detect corresponding specific immune among the patient that FATEmRNA expresses and replys and have at cancerous tissue.At part cancer tissues expressed FATEmRNA, but serum do not have in the tumour patient of FATE antibody, still can detect the immunne response of CD8+T cell to tumour antigen FATE and antigen peptide.This experiment provides foundation for experiment in the clinical body of further using tumour antigen FATE and antigen peptide treatment tumour.
The preparation of the tumor antigen protein matter of embodiment 9, sudden change and serum antibody screening thereof
Use the DNA that well-known method has prepared following random mutation, the 534th base in the sequence number 2 sported G by T, also just the 141st amino acid in the sequence number 1 is sported R by L thus; The 552nd base in the sequence number 2 sported C by G, also just the 147th amino acid in the sequence number 1 is sported P by R thus.The dna clone of sudden change in expression vector, is expressed the tumour antigen of sudden change, in detail with reference to embodiment 4.Use the serum of the tumour antigen of the sudden change for preparing, find in the serum of tumour patient, also to exist the antibody of these sudden change tumour antigens with reference to embodiment 5 screening tumour patients.Because the mutational site among this embodiment is selected at random, also there is the antibody of the tumor antigen protein matter that produces at other site mutation in this explanation in the serum of tumour patient.This embodiment illustrates that the tumour antigen of sudden change also has the induced tumor patient to produce immunne response, repels the ability of killing tumor cell, and the potential of the clinical immunotherapy of the tumour of being applied to is arranged.
Use tumor antigen protein matter of the present invention and tumor antigen peptide the medicine of activation antineoplastic immune can be provided, diagnostic method of tumors can be provided.The tumor antigen protein matter among anti-the present invention or the antibody of tumor antigen peptide can be used for the preparation of affinity chromatography, cDNA library screening, immunology diagnosis or medicine.
Sequence table comprises sequence 1 and sequence 2
Sequence length: 183
Topology: linearity
Sequence kind: peptide
The source:
Biological name: people (Homo sapiens)
Tissue types: liver cancer tissue
Sequence signature:
The mark of feature: peptide
Location: 1..183
Sequence 1:
Met?Ala?Gly?Gly?Pro?Pro?Asn?Thr?Lys?Ala?Glu?Met?Glu?Met?Ser?Leu
1 5 10 15
Ala?Glu?Glu?Leu?Asn?His?Gly?Arg?Gln?Gly?Glu?Asn?Gln?Glu?His?Leu
20 25 30
Val?Ile?Ala?Glu?Met?Met?Glu?Leu?Gly?Ser?Arg?Ser?Arg?Gly?Ala?Ser
35 40 45
Gln?Lys?Lys?Gln?Lys?Leu?Glu?Gln?Lys?Ala?Ala?Gly?Ser?Ala?Ser?Ala
50 55 60
Lys?Arg?Val?Trp?Asn?Met?Thr?Ala?Thr?Arg?Pro?Lys?Lys?Met?Gly?Ser
65 70 75 80
Gln?Leu?Pro?Lys?Pro?Arg?Met?Leu?Arg?Glu?Ser?Gly?His?Gly?Asp?Ala
85 90 95
His?Leu?Gln?Glu?Tyr?Ala?Gly?Asn?Phe?Gln?Gly?Ile?Arg?Phe?His?Tyr
100 105 110
Asp?Arg?Asn?Pro?Gly?Thr?Asp?Ala?Val?Ala?Gln?Thr?Ser?Leu?Glu?Glu
115 120 125
Phe?Asn?Val?Leu?Glu?Met?Glu?Val?Met?Arg?Arg?Gln?Leu?Tyr?Ala?Val
130 135 140
Asn?Arg?Arg?Leu?Arg?Ala?Leu?Glu?Glu?Gln?Gly?Ala?Thr?Trp?Arg?His
145 150 155 160
Arg?Glu?Thr?Leu?Ile?Ile?Ala?Val?Leu?Val?Ser?Ala?Ser?Ile?Ala?Asn
165 170 175
Leu?Trp?Leu?Trp?Met?Asn?Gln
180
Sequence length: 1137
Chain: two strands
Topology: linearity
Sequence kind: cDNA to mRNA
Hypothetical sequence: do not have
Antisense: do not have
Origin:
Biological name: people (Homo sapiens)
Tissue types: liver cancer tissue
Sequence signature:
The expression mark of feature: 5 ' UTR
Location: 1..112
The expression mark of feature: CDS
Location: 113..664
The expression mark of feature: 3 ' UTR
Location: 665..1095
The expression mark of feature: polyA site
Location: 1096..1137
Sequence 2:
AGAGGATCCC?AATTTAGCTG?CGCACAGGGA?GGTGATTTTC?TGAGTGTGAC?TCCTCTGTTC 60
CTGGCACCCT?GTGCATCCTT?AGCCATAGCT?TACAAGAGAA?CAGCTGGTTG?TGATGGCAGG 120
AGGCCCTCCC?AACACCAAGG?CGGAGATGGA?AATGTCCCTG?GCAGAAGAAC?TGAATCATGG 180
ACGCCAAGGG?GAAAACCAAG?AGCACCTGGT?GATAGCAGAA?ATGATGGAGC?TTGGATCTCG 240
GTCCCGGGGT?GCCTCCCAGA?AGAAGCAGAA?GTTGGAACAA?AAAGCTGCTG?GCTCTGCTTC 300
AGCCAAACGA?GTTTGGAATA?TGACTGCCAC?CCGACCCAAG?AAAATGGGGT?CCCAGCTGCC 360
AAAGCCCAGA?ATGCTGAGAG?AATCAGGCCA?TGGGGATGCC?CATCTCCAGG?AGTACGCTGG 420
CAATTTCCAA?GGCATACGTT?TCCATTATGA?TCGCAACCCA?GGGACAGATG?CAGTGGCGCA 480
GACTAGCCTG?GAAGAGTTCA?ATGTACTGGA?GATGGAAGTC?ATGAGAAGAC?AGCTGTATGC 540
AGTCAACCGG?CGTCTGCGCG?CCCTGGAGGA?ACAGGGCGCC?ACCTGGCGCC?ACAGGGAGAC 600
CCTGATCATC?GCCGTGCTGG?TGTCGGCCAG?CATTGCCAAC?CTGTGGCTGT?GGATGAACCA 660
GTGATCGCCC?CAGCGCGGCC?TCCGTATTGG?AGCCCTCCCT?GCTTCCCCTT?CTTTCTTTCC 720
TCTTTCCCCA?GGCCGCCACT?GCCCTTGCCC?CTTTCATCTC?CCAGCAGCCC?TCAGGAGCGT 780
CAGGATCATT?TTCAACTCTG?GTTAGGCCTC?CTACCTGGGG?AGGCCAGGTC?ACTGCACTGG 840
GAGGTCCTGG?CTGCTGCGAA?GCTGGAGGAG?GACTGCGTGG?GCTGAGATGC?CACCCTTTGA 900
AGGGTGAACA?GCATGGCGGC?ATCTGGGCCC?CACAGTAACA?CCTAGTGGCA?ACCTTGCCTT 960
CCTGACCTCA?GCGGCCCTTC?TGTTCCATCC?TCTGTGGGCA?GGGGTGTGGC?TTTGTTTTCC 1020
TCCCTCGTTT?GCTTCCACCT?CGTGCACAGC?GCTCTGCACA?GACAACACGC?TCAATAAAAG 1080
TTCAGCCATA?GCAGCAAAAA?AAAAAAAAAA?AAAAAAAAAA?AAAAAAAAAA?AAAAAAA 1137
PIO34447-sequence?list.txt
SEQUENCE?LISTING
＜110〉Peking University
＜120〉a kind of tumor antigen protein matter and tumor antigen peptide
<130>PIO34447
<160>2
<170>PatentIn?version?3.1
<210>I
<211>183
<212>PRT
<213>Homo?sapiens
<400>1
Met?Ala?Gly?Gly?Pro?Pro?Asn?Thr?Lys?Ala?Glu?Met?Glu?Met?Ser?Leu
1 5 10 15
Ala?Glu?Glu?Leu?Asn?His?Gly?Arg?Gln?Gly?Glu?Asn?Gln?Glu?His?Leu
20 25 30
Val?Ile?Ala?Glu?Met?Met?Glu?Leu?Gly?Ser?Arg?Ser?Arg?Gly?Ala?Ser
35 40 45
Gln?Lys?Lys?Gln?Lys?Leu?Glu?Gln?Lys?Ala?Ala?Gly?Ser?Ala?Ser?Ala
50 55 60
Lys?Arg?Val?Trp?Asn?Met?Thr?Ala?Thr?Arg?Pro?Lys?Lys?Met?Gly?Ser
65 70 75 80
Gln?Leu?Pro?Lys?Pro?Arg?Met?Leu?Arg?Glu?Set?Gly?His?Gly?Asp?Ala
85 90 95
His?Leu?Gln?Glu?Tyr?Ala?Gly?Asn?Phe?Gln?Gly?Ile?Arg?Phe?His?Tyr
100 105 110
Asp?Arg?Asn?Pro?Gly?Thr?Asp?Ala?Val?Ala?Gln?Thr?Set?Leu?Glu?Glu
115 120 125
Phe?Asn?Val?Leu?Glu?Met?Glu?Val?Met?Arg?Arg?Gln?Leu?Tyr?Ala?Val
130 135 140
Asn?Arg?Arg?Leu?Arg?Ala?Leu?Glu?Glu?Gln?Gly?Ala?Thr?Trp?Arg?His
145 150 155 160
Arg?Glu?Thr?Leu?Ile?Ile?Ala?Val?Leu?Val?Ser?Ala?Ser?Ile?Ala?Asn
165 170 175
Leu?Trp?Leu?Trp?Met?Asn?Gln
180
<210>2
<211>1137
<212>DNA
<213>Homo?sapiens
<400>2
agaggatccc?aatttagctg?cgcacaggga?ggtgattttc?tgagtgtgac?tcctctgttc 60
PIO34447-sequence?list.txt
ctggcaccct?gtgcatcctt?agccatagct?tacaagagaa?cagctggttg?tgatggcagg 120
aggccctccc?aacaccaagg?cggagatgga?aatgtccctg?gcagaagaac?tgaatcatgg 180
acgccaaggg?gaaaaccaag?agcacctggt?gatagcagaa?atgatggagc?ttggatctcg 240
gtcccggggt?gcctcccaga?agaagcagaa?gttggaacaa?aaagctgctg?gctctgcttc 300
agccaaacga?gtttggaata?tgactgccac?ccgacccaag?aaaatggggt?cccagctgcc 360
aaagcccaga?atgctgagag?aatcaggcca?tggggatgcc?catctccagg?agtacgctgg 420
caatttccaa?ggcatacgtt?tccattatga?tcgcaaccca?gggacagatg?cagtggcgca 480
gactagcctg?gaagagttca?atgtactgga?gatggaagtc?atgagaagac?agctgtatgc 540
agtcaaccgg?cgtctgcgcg?ccctggagga?acagggcgcc?acctggcgcc?acagggagac 600
cctgatcatc?gccgtgctgg?tgtcggccag?cattgccaac?ctgtggctgt?ggatgaacca 660
gtgatcgccc?cagcgcggcc?tccgtattgg?agccctccct?gcttcccctt?ctttctttcc 720
tctttcccca?ggccgccact?gcccttgccc?ctttcatctc?ccagcagccc?tcaggagcgt 780
caggatcatt?ttcaactctg?gttaggcctc?ctacctgggg?aggccaggtc?actgcactgg 840
gaggtcctgg?ctgctgcgaa?gctggaggag?gactgcgtgg?gctgagatgc?caccctttga 900
agggtgaaca?gcatggcggc?atctgggccc?cacagtaaca?cctagtggca?accttgcctt 960
cctgacctca?gcggcccttc?tgttccatcc?tctgtgggca?ggggtgtggc?tttgttttcc 1020
tccctcgttt?gcttccacct?cgtgcacagc?gctctgcaca?gacaacacgc?tcaataaaag 1080
ttcagccata?gcagcaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaa 1137
Claims (2)
1, the application of a kind of tumor antigen protein matter in preparation treatment liver-cancer medicine, the aminoacid sequence of described tumor antigen protein matter is shown in sequence 1, this protein is by decomposing, can produce the peptide fragment that combines with major histocompatibility complex (MHC)-I quasi-molecule, can be discerned by the T cell at bonding state in the cell.
2, the application of a kind of tumor antigen peptide in preparation treatment liver-cancer medicine, the aminoacid sequence of described tumor antigen peptide and has the peptide fragment same function that is produced with the described tumor antigen protein matter of claim 1 shown in the sequence 1 the 140th～the 148th.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB03136411XA CN1326875C (en) | 2003-05-15 | 2003-05-15 | Tumor antigen protein and tumor antigen peptide |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB03136411XA CN1326875C (en) | 2003-05-15 | 2003-05-15 | Tumor antigen protein and tumor antigen peptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1548457A CN1548457A (en) | 2004-11-24 |
| CN1326875C true CN1326875C (en) | 2007-07-18 |
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|---|---|---|---|
| CNB03136411XA Expired - Fee Related CN1326875C (en) | 2003-05-15 | 2003-05-15 | Tumor antigen protein and tumor antigen peptide |
Country Status (1)
| Country | Link |
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| CN (1) | CN1326875C (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1227602A (en) * | 1996-06-07 | 1999-09-01 | 伊东恭悟 | Tumor antigen proteins, genes thereof and tumor antigen peptides |
-
2003
- 2003-05-15 CN CNB03136411XA patent/CN1326875C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1227602A (en) * | 1996-06-07 | 1999-09-01 | 伊东恭悟 | Tumor antigen proteins, genes thereof and tumor antigen peptides |
Non-Patent Citations (3)
| Title |
|---|
| BJ-HCC-2, a tumor-associated gene which is expressed onlyin the normal pancreas and testis Dong,X.和Chen,W,NCBI中人序列AY101182 2003 * |
| BJ-HCC-2, a tumor-associated gene which is expressed onlyin the normal pancreas and testis Dong,X.和Chen,W,NCBI中人序列AY101182 2003;BJ-HCC-2, a tumor-associated gene which is expressed onlyinthe normal pancreas and testis Dong,X.和Chen,W,NCBI中人序列AAM48744 2003 * |
| BJ-HCC-2, a tumor-associated gene which is expressed onlyinthe normal pancreas and testis Dong,X.和Chen,W,NCBI中人序列AAM48744 2003 * |
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| Publication number | Publication date |
|---|---|
| CN1548457A (en) | 2004-11-24 |
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