CN1548457A - A kind of tumor antigen protein and tumor antigen peptide - Google Patents

Abstract

The present invention relates to a tumor antigen protein and tumor antigen peptide. The tumor antigen protein can be decomposed in the cell to produce a peptide fragment that combines with the major histocompatibility complex (MHC)-I class molecule and can be recognized by T cells in the combined state. The DNA sequence encoding the tumor antigen protein has the nucleotide sequence of the accession number in the gene library: FATE AF249872. The tumor antigen protein/tumor antigen peptide can be used to prepare medicines for treating liver cancer or colon cancer.

Description

A kind of tumor antigen protein and tumor antigen peptide

technical field

The invention relates to a tumor antigen protein and a tumor antigen peptide, in particular to a tumor antigen protein and a tumor antigen peptide for treating liver cancer and intestinal cancer.

technical background

Malignant tumors are a large class of diseases that threaten human health. Tumor occurrence is a very complex process. An important factor for the occurrence and progression of tumor cells is that they can evade the surveillance of the body's immune system. Therefore, how to stimulate and evoke the body's immune system to recognize and reject tumor cells with "non-self" components is a key factor in finding new tumor treatments. breakthrough. In the initial stage of immunotherapy research on tumors, people have developed many methods to stimulate the body's immune system to reject tumor cells with "non-self" components. Although these methods have achieved good results in the treatment of animal tumor models, but There is still a lack of significant effects in the treatment of human tumors.

It is known that the immune system in the human body, including humoral immunity and cellular immunity, especially T cell immunity, plays an important role in killing and inhibiting tumors. With the understanding of the nature of the immune response, people found that no matter how complicated the process of the immune response is, what ultimately leads to the activation of T cells and the killing effect is to combine with the major histocompatibility complex MHC to form antigen peptide complexes and co- Stimulatory signal, that is, cytotoxic T cells (CTL) recognize tumor antigen proteins/peptides and major histocompatibility complex (MHC) class I antigens expressed on the surface of tumor cells through T cell receptors (TCR) on their own surface Combined to form a complex to attack and kill tumor cells.

The production of tumor antigen protein/peptide is first expressed by tumor cells into tumor antigens, which are decomposed into antigen peptides of 8-10 amino acid sizes by various proteases in the cells, and these antigen peptides are produced together with MHC I in the endoplasmic reticulum These molecules are combined with molecules, expressed on the surface of cells through the Golgi complex, and finally presented to tumor antigen-specific CTLs, thereby inducing an immune response and killing and clearing tumor cells. This protein antigen molecule that exists in tumor cells and can bind to MHC molecules and induce the body's immune response is a tumor antigen. These antigens can be used to induce the body to produce an immune response that only targets tumor cells, thereby specifically killing tumor cells in the body to achieve the effect of treating or preventing tumor cell metastasis. Therefore, in recent decades, people have focused on the search for tumor-specific and/or related antigens in the treatment of tumors with immune methods.

In the 1950s, Foley et al. immunized mice with chemically induced tumors with inactivated autologous tumor cells, and then inoculated the mice with live tumor cells, which were rejected and unable to grow In contrast, mice that were not immunized with inactivated autotumor cells were inoculated with live tumor cells, the tumor cells were not rejected and grew into tumors, and the mice died. This experiment proved convincingly that there are tumor-specific antigens in chemically induced tumors, but this protective response has precise specificity, that is, it is only against the same type of tumor and not against different types of tumors, even for Different types of tumors induced in the same mouse with the same carcinogen were also not protective.

In 1991, T. Boon et al. identified the tumor antigen protein from human melanoma cells for the first time and named it MAGE (Science, 254:1643-1647, 1991). Subsequently, other tumor antigen proteins were identified, which can be divided into five categories: tumor-testis (CT) antigen protein, differentiation antigen protein, mutated tumor antigen protein, overexpressed tumor antigen protein and virus antigen protein. Among them, tumor-testis (CT) antigen protein is the most widely used clinically.

Tumor-testis (CT) antigen proteins are the most identified class of tumor antigens, and their encoding genes are characterized by their expression in many types of tumors, such as melanoma, lung cancer, sarcoma and bladder cancer It is expressed, but it is not expressed in normal tissues except the testis, and there is a certain amount of expression in the placenta, ovary, and pancreas. Because the testis is an immune privileged site, this type of antigen is considered to be a tumor-specific antigen in therapy, and it is the most promising type of antigen for tumor immunotherapy. At present, this type of antigen is mainly used clinically, such as MAGE-1 and NY-ESO-1, which have a high positive rate in certain types of tumors and have good immunogenicity. , has a good prospect when used in tumor immunotherapy.

So far, the most representative methods for identifying tumor antigen proteins are: library screening based on specific cellular immune response, cDNA expression library screening based on humoral immune response, combinatorial peptide library screening, pickling off method and bioinformatics approach.

Liver cancer and intestinal cancer are the most common and malignant tumors among human tumors, especially in recent years, the incidence rate has gradually increased. Due to the high degree of malignancy and rapid growth of liver cancer, once it is discovered, it is already in the middle and late stage. At present, the treatment of liver cancer is mainly local treatment methods such as traditional surgical resection and radiotherapy. However, since most patients have entered the middle and late stage when they are diagnosed, And liver cancer is quite resistant to radiotherapy and chemotherapy. Therefore, the treatment effect and prognosis are often poor, and the one-year and five-year survival rates of liver cancer patients are very low compared with the survival rates of other types of tumor patients. Finding breakthroughs in treatment methods or new treatment methods has always been the goal pursued by clinical oncologists. People are looking forward to new methods to improve the early diagnosis of liver cancer and the good prognosis of patients, as well as to remove residual tumor cells in the body after surgery. and prevent tumor cell metastasis.

Contents of the invention

The purpose of the present invention is to provide a tumor antigen protein and tumor antigen peptide which can be used for the treatment of liver cancer and colon cancer.

The purpose of the present invention is achieved through the following technical solutions:

The present invention provides a tumor antigen protein, which can produce a peptide fragment combined with major histocompatibility complex (MHC)-I class molecules and recognized by T cells in the combined state through intracellular decomposition of the protein, It has the amino acid sequence described in (a) or (b) below:

a) has the amino acid sequence shown in Sequence 1;

b) Substituting one or several amino acid residues in the amino acid sequence in (a), deleting or adding mutations to produce a derivative protein, and the derivative protein has the same function as the protein in (a).

The present invention provides a DNA sequence, which encodes said tumor antigen protein.

The DNA sequence of the present invention has the nucleotide sequence shown in Sequence 2, and its accession number in the gene library is: FATE AF249872.

The invention provides the application of said protein as tumor-testis antigen.

The invention provides the application of the gene as tumor-testis antigen gene.

The present invention provides a tumor antigen peptide having the same function as the peptide fragment produced by the tumor antigen protein, which has the amino acid sequence of the 140th to 148th amino acid sequence in the sequence 1.

The application of the tumor antigen protein of the present invention in the preparation of medicines for treating liver cancer.

The application of the tumor antigen protein of the present invention in the preparation of medicine for treating intestinal cancer.

The application of the tumor antigen peptide of the present invention in the preparation of drugs for treating liver cancer.

The application of the tumor antigen peptide of the present invention in the preparation of drugs for treating liver cancer.

The advantages of the tumor antigen protein and tumor antigen peptide provided by the present invention are: the present invention identifies the FATE gene as a tumor-testis antigen gene for the first time; when using immune methods to treat cancer, these antigens can be used to induce the body to produce The immune response of cells can specifically kill tumor cells in the body while not killing normal cells to achieve the effect of treating or preventing tumor cell metastasis; at the same time, the occurrence of tumor can be detected by detecting the tumor antigen gene or antibody. , development and whether there is metastasis for diagnosis.

Description of drawings

Fig. 1 is the RT-PCR detection result of the gene FATE encoding the antigenic protein of the present invention in 16 kinds of adult normal tissues; 1～16 in the figure represent respectively adult normal tissues spleen, prostate, ovary, small intestine, large intestine, leukocyte, testis, heart , lung, liver, brain, kidney, placenta, skeletal muscle, pancreas, thymus;

Fig. 2 is the RT-PCR result of the gene FATE encoding the antigenic protein of the present invention in the cancer tissue of liver cancer and the paired paracancerous tissue; ca in the figure represents the cancerous tissue, and adj represents the paired paracancerous tissue;

Fig. 3 is the Northern blot result of the gene FATE encoding the antigenic protein of the present invention in liver cancer tissue and paired paracancerous tissue; ca in the figure represents cancerous tissue, and adj represents paired paracancerous tissue;

Figure 4 is a representative result of the reaction of the recombinant protein of the antigenic protein of the present invention in Escherichia coli and the serum of liver cancer patients detected by Western blot method; M in the figure represents the protein molecular weight standard reference, and 1 represents the expression of the tumor in the present invention- The result of the reaction of the Escherichia coli lysate of the testis antigen recombinant protein with the monoclonal antibody against the histidine tag, 2 represents the result of the reaction of the purified tumor-testis antigen recombinant protein of the present invention with the monoclonal antibody against the histidine tag , 3 represents the result of the reaction of the Escherichia coli lysate expressing the tumor-testis antigen recombinant protein of the present invention with the serum of liver cancer patients, and 4 represents the result of the reaction of the purified tumor-testis antigen recombinant protein of the present invention with the serum of liver cancer patients, 5 represents the result of the reaction between the E. coli lysate transfected with an empty expression vector and the serum of liver cancer patients, and 6 represents the result of the reaction between the E. coli lysate transfected with an irrelevant protein control expression vector and the serum of liver cancer patients;

分别代表重组蛋白与3个不同肿瘤病人血清反应的结果。Fig. 5 is the result of the reaction of the tumor-testis antigen protein recombinant protein in Escherichia coli of the present invention and the serum of liver cancer patients detected by ELISA method; wherein the X-axis represents the dilution of serum, and the Y-axis represents the absorbance when the wavelength is 410nm ;

Respectively represent the results of the recombinant protein reacting with sera from three different tumor patients.

Detailed ways

Embodiment 1, the screening of FATE gene

Using the testis-specific expression as the subject term, searched the gene database (GenBank) and the literature search database (PubMed) of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) in the United States for those that have been rigorously expressed. Defined testis-specific expression of genes in normal human tissues, but it must not have any tumor-associated reports so far. From this, we obtained several genes specifically expressed in human normal tissues including the FATE gene, and took them as the target genes for our next Unigene and SAGE database analysis.

Embodiment 2, Unigene and SAGE database analysis

Unigene is a web tool that automatically classifies all Genbank sequences derived from the same gene. In the Unigene database, each Unigene gene contains sequences derived from various tissues but belonging to the same gene fragments with different sizes, and the tissue sources and expression information of these sequences are annotated. The SAGE (Serial Analysis of Gene Expression) database is an online tool that can quantify and qualify the expression profile of a specific gene. Therefore, by using the Unigene and SAGE databases, the temporal and spatial expression of a specific gene can be analyzed to obtain its related functional information. In the process of the present invention, three criteria are used to screen candidate genes to expect to obtain the tumor-testis (CT) antigen gene of purpose: 1. the candidate gene must be a strictly defined gene specifically expressed in human normal tissue, but It must not have any tumor-associated reports so far. 2. In the Unigene database analysis in the process of the present invention, the Unigene data of the candidate gene must contain the expressed sequence tag (EST) derived from the tumor. 3. In the SAGE database analysis in the process of the present invention, if there is a SAGE label in the candidate gene, only SAGE tags that strictly correspond to candidate genes can be used in the analysis of candidate genes. When the above three criteria were adopted, the candidate gene FATE was considered as the most likely tumor-testis antigen gene. In the Unigene data of the FATE gene, there are a total of 33 EST fragments, although 31 of them are derived from testicular tissue, but the other 2 are derived from tumor tissue; in the SAGE data of the FATE gene, no FATE gene is derived from tumor The SAGE label for the organization. Based on the above analysis, it is considered that the FATE gene may be the target gene in the present invention—the tumor-testis antigen gene, and the next step will be to design experiments to prove it.

Embodiment 3, analyze the expression of FATE gene with the method for RT-PCR

cDNA of 16 commercial human normal tissues from the biotechnology company CLONTECH (spleen, prostate, testis, ovary, small intestine, large intestine, white blood cells, heart, lung, liver, brain, kidney, pancreas, placenta, skeletal muscle, thymus), cDNAs from cancer tissues and matched paracancerous tissues of 62 liver cancer patients, cDNAs from cancer tissues and matched paracancerous tissues of 14 lung cancer patients, and cancer tissues and matched paracancerous tissues of 14 gastric cancer patients Tissue cDNA and cDNA from 14 colorectal cancer patients' cancer tissues and paired paracancerous tissues were used to detect the expression of FATE gene in normal tissues and tumor tissues.

The conditions for RT-PCR in the present invention: use the advanced hot-start DNA polymerase of CLONTECH Company, 94°C, 15 seconds; 68°C, 30 seconds; 72°C, 30 seconds, 30 cycles, and then, 72°C, 6 minutes. The primer sequence for RT-PCR, forward primer: ctg ttc ctg gca ccc tgt gca tcc; reverse primer: gat gcc gcc atgctg ttc acc c. As a result, it was found that the FATE gene was expressed in 66% of liver cancer specimens and 21% of intestinal cancers, but not in the paracancerous tissues paired with it (as shown in Figure 2); in 16 important normal tissues of adults, it was found The FATE gene is also expressed in the pancreas, and its expression level is about 1/5 of the expression level in the testis tissue (as shown in Figure 1). The testes are about twice as low. Based on the above experimental data, this example proves that the FATE gene is a new tumor-testis antigen gene consistent with the analysis results of Unigene and SAGE database in Example 1.

Example 4. Analysis of the expression of FATE gene in liver cancer and intestinal cancer by Northern hybridization

Using TRIZOL reagent (PROMEGA) to extract total RNA from cancer tissues and paired paracancerous tissues of patients with liver cancer and colon cancer, and then use mRNA isolation kit (PROMEGA) to isolate mRNA from total RNA. In the presence of formamide and formaldehyde, 2ug of mRNA was denatured, after agarose electrophoresis, it was transferred to Hybond-N+ nylon membrane (Amersham), and fixed by ultraviolet crosslinking. The DNA fragments amplified in Example 2 were labeled with Digoxigenin DNA Probe Labeling Kit (Roche) to make DNA probes, hybridized with the mRNA on the membrane according to the conventional Northern hybridization method, and then carried out autoradiography, The mRNA of the testis-tumor antigen protein gene FATE in the present invention was detected. Then, boil the membrane used to detect the mRNA of the gene in 1% SDS solution, remove the digoxin-labeled probe, use the labeled housekeeping gene G3PDH as a probe, and use the same method to perform Northern hybridization to detect mRNA was used as a positive control. The result is shown in Figure 3. It can be seen from this result that the mRNA of the tumor antigen protein gene of the present invention is only expressed in cancer tissues, but not in the paired paracancerous tissues. It is consistent with the characteristics of the tumor-testis antigen gene.

Embodiment 5, expression of FATE gene recombinant protein in escherichia coli

Using the pQE30 prokaryotic expression vector (INVOTRIGEN), IPTG induced the expression of the recombinant protein for 6 hours in Escherichia coli strain M15 (INVOTRIGEN) under the growth condition of 37°C. The expression of recombinant protein was identified by SDS-PAGE protein electrophoresis. Afterwards, the recombinant protein was separated and purified from the whole protein of E. coli using a nickel ion affinity chromatography column (INVOTRIGEN). The recombinant protein was identified by using the monoclonal antibody against the 6 histidines contained in the recombinant protein by Western hybridization. The plasmid pQE30-FATE expressing the tumor-testis antigen FATE and the Escherichia coli strain M15 containing the plasmid pQE30-FATE were stored in a -80°C low-temperature refrigerator in the T-cell Research Laboratory of the Department of Immunology, School of Basic Medicine, Peking University Health Science Center of the People's Republic of China. for May 26, 2002.

Embodiment 6, detect the anti-FATE recombinant protein antibody in the serum of patients with liver cancer and intestinal cancer by Western hybridization and ELISA

With reference to the method in Journal of Experimental Medicine, 187:1349, 1998, the serum of tumor patients with positive FATE mRNA transcripts was screened, and it was found that there were antibodies against FATE recombinant protein in the serum of tumor patients (Fig. 4, 5). Therefore, FATE is a new tumor-testis antigen, which has the ability to induce immune response in tumor patients, reject and kill tumor cells, and has the potential to be applied to clinical immunotherapy of tumors.

Example 7. Application of Tumor Antigen Peptides to Induce CTL Formation in Vitro and CTL Killing Experiments in Vitro

Refer to the experimental method in Chinese Journal of General Surgery (17: 218, 2002). Culture 2ml of PBMC (peripheral blood mononuclear cells) at a concentration of 2× ^{10 6 /} ml in a 24-well culture plate as effector lymphocytes. Add 40ug/ml of tumor antigen peptide having the amino acid sequence of 140th to 148th in Sequence 1, incubate at 37°C for 2 hours, irradiate with 30GY ⁶⁰ Co gamma rays, centrifuge, wash away the remaining peptide, and use as Antigen-extracting cells, according to the number of APC cells with effector cells:APC ratio of 1.3:1, add antigen-extracting cells to the above-mentioned effector cells for co-culture, and add IL-2 (provided by Ludwig Institute for Cancer Research, Australia) 25U/ml after 24 hours to continue nourish. Afterwards, every 7 days, antigen-extracting cells were added to effector cells according to the number of antigen-extracting cells at the effector cell:antigen-extracting cell ratio of 1.3:1, and the effector cells were stimulated three times. The killing effect of CTL was detected on the 28th day. The killing experiment was carried out as follows: (1) Using a non-radioactive cytotoxicity assay box, measuring the LDH release value after CTL and target cells were co-cultured to detect the killing effect of CTL. Operate according to the recommended procedure of the kit. The medium used was phenol red-free RPMI 1640 containing 3% FCS. Three replicate wells were set up for each reaction, and the spontaneous release value of target cells, the maximum release value of target cells, the spontaneous release value of effector cells, the volume correction value and Medium background value. The following formula was used to calculate the killing effect: killing effect (%)=(experimental value-effector cell spontaneous release value-target cell spontaneous release value)/(target cell maximum release value-target cell spontaneous release value)×100. (2) After the mixed lymphocytes and target cells were co-cultured for 4 hours, the morphological changes of lymphocytes and target cells were observed. After 28 days, peripheral blood mononuclear cells expanded 32-fold, among which the proportion of CD3 positive cells increased by 16% (from 54% to 70%), and the proportion of CTL increased by 20% (from 36% to 56%). When the ratio of effector cells: target cells was 10:1, the killing effect of CTL on autologous lymphoblastoid cells incubated with tumor antigen peptide (synthesized by Shanghai Gil Biochemical Co., Ltd.) was 62.5%, and the killing effect on tumor cells positive for tumor antigen FATE The effect was 40.2%, both of which were significantly higher than the killing effect on autologous lymphoblastoid cells (17.9%) and the killing effect on tumor cells negative for tumor antigen FATE (1.6%); when the ratio of effector cells: target cells was 3.3:1 , the killing effect of CTL on autologous lymphoblasts incubated with tumor antigen peptide was 53.6%, which was significantly higher than the killing effect on autologous lymphoblasts (15.6%). These results indicate that the tumor antigen peptide of the tumor antigen FATE can effectively induce CTLs with specific killing tumor cells from the PBMC of tumor patients, thereby enhancing the ability of tumor patients to clear tumors.

Example 8. Detection of IFNγ-secreting ability of CTL cells stimulated by tumor antigen peptides

Refer to the experimental method in Chinese Medical Journal (81: 1234, 2001). Quantification of IFN-γ was performed by enzyme-linked immunospot assay (ELISPOT). 96-well flat-bottomed nitrocellulose plate (purchased from Millipore, France), coated with anti-human IFNγ monoclonal antibody (dissolved in pH 9.6 carbonate buffer, final concentration 2ug/ml), overnight at 4°C; After washing with phosphate buffered saline (PBS) containing 0.05% Tween 20, block with RPMI 1640 culture solution containing 10% AB serum for 1 hour at room temperature in the dark, then wash with 0.05% Tween PBS, and add 7-day-induced Effector cells (Effector cells, E) 5×10 ⁴ per well. Afterwards, the experiment was divided into two groups, that is, the effector cell and T2 cell group (E+T2), and the effector cell and T2 cell loading tumor antigen peptide group (E+T2 tumor antigen peptide), and each group made duplicate wells. Pure T2 cells and T2 cells loaded with 10ug/ml tumor antigen peptide were incubated in serum-free RPMI 1640 for 2 hours at 37°C and 5% CO ₂ , irradiated with ⁶⁰ Coγ rays 100GY, and washed with serum-free RPMI 1640 to remove excess After the antigen peptide was used as stimulator cells, 1×10 ⁵ cells/well were added to each effector cell well; the reaction system was incubated in RPMI 1640 culture medium without cytokines and serum at 37°C and 5% CO ₂ for 18 ~20 hours later, wash the culture plate with 0.05% TweenPBS to remove residual cells; then add 0.2ug/ml biotin-labeled anti-human IFNγ secondary antibody, incubate at 37°C for 2 hours, wash the plate; add alkaline phosphatase labeling Streptavidin 1ug/m, incubate at room temperature for 1 hour in the dark; wash the plate, add the substrate chromogenic solution, develop the color in the dark for 5 minutes, rinse with tap water, and terminate the reaction. After the reaction plate was dried, count the number of black-purple spots in each well under a dissecting microscope, and calculate the average value of the duplicate wells. The number of spots in the E+T2 tumor antigen peptide group minus the number of spots in the E+T2 group was the frequency of tumor antigen-specific CTL cells. In ELISPOT detection, using the frequency of IFNγ-producing CD8 ⁺ T cells as an index, the responders to the tumor antigen FATE were detected in patients whose tumor tissue expressed FATE mRNA, but were not detected in patients with no FATE mRNA expression in cancer tissue to the corresponding specific immune response. In tumor patients whose tumor tissues express FATE mRNA but have no FATE antibody in their serum, the immune response of CD8+ T cells to tumor antigen FATE and antigenic peptide can still be detected. This experiment provides a basis for the clinical in vivo experiment of further application of tumor antigen FATE and antigen peptide to treat tumors.

Example 9. Preparation of mutated tumor antigen protein and its serum antibody screening

The following randomly mutated DNA was prepared by using well-known methods, the 534th base in SEQ ID NO: 2 was mutated from T to G, thus the 141st amino acid in SEQ ID NO: 1 was mutated from L to R; The 552nd base in SEQ ID NO: 2 was mutated from G to C, thus the 147th amino acid in SEQ ID NO: 1 was mutated from R to P. Cloning the mutated DNA into an expression vector to express the mutated tumor antigen, refer to Example 4 for details. The prepared mutated tumor antigens were used to screen the serum of tumor patients with reference to Example 5, and it was found that antibodies to these mutated tumor antigens also existed in the serum of tumor patients. Since the mutation sites in this example are randomly selected, this shows that antibodies against tumor antigen proteins produced by mutations at other sites also exist in the serum of tumor patients. This example shows that the mutated tumor antigen also has the ability to induce tumor patients to generate an immune response, to reject and kill tumor cells, and has the potential to be applied to clinical immunotherapy of tumors.

Using the tumor antigen protein and tumor antigen peptide of the present invention can provide medicines for activating anti-tumor immunity and methods for diagnosing tumors. The antibody against the tumor antigen protein or tumor antigen peptide of the present invention can be used in affinity chromatography, screening of cDNA library, immunological diagnosis or preparation of medicine.

The sequence listing includes sequence 1 and sequence 2

Sequence 1 is the amino acid sequence of tumor-testis antigen protein FATE:

Sequence length: 183

Topology: Linear

Sequence Type: Peptide

source:

Biological name: Human (Homo sapiens)

Tissue Type: Liver Cancer Tissue

Sequence features:

Signature of feature: Peptide

Existence 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

                           

Sequence 2 is the nucleotide sequence of tumor-testis antigen protein gene FATE:

Sequence length: 1137

Chain type: double chain

Topology: Linear

Sequence type: cDNA to mRNA

Hypothetical sequence: none

Antonym: None

origin:

Biological name: Human (Homo sapiens)

Tissue Type: Liver Cancer Tissue

Sequence features:

Characteristic notation: 5'UTR

Existence location: 1..112

Characteristic notation: CDS

Existence location: 113..664

Characteristic notation: 3’UTR

Existence location: 665..1095

Characteristic notation: polyA site

Existence 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 CATTCTCCAGG 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 AGCCCCTCCCT 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

SEQUENCE LISTING

<110> Peking University

<120> A tumor antigen protein and tumor antigen peptide

<130>PI034447

<160>2

<170>PatentIn version 3.1

<210>1

<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 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

<210>2

<211>1137

<212>DNA

<213>Homo sapiens

<400>2

agaggatccc aatttagctg cgcacaggga ggtgattttc tgagtgtgac tcctctgttc 60

ctggcaccct gtgcatccctt 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 (10)

1. A tumor antigen protein, which can produce peptide fragments combined with major histocompatibility complex (MHC)-I class molecules and recognized by T cells in the combined state through intracellular decomposition, which has An amino acid sequence as described in (a) or (b) below: (a) having the amino acid sequence shown in Sequence 1; (b) The amino acid sequence in (a) undergoes one or several amino acid residue substitutions, deletion or addition mutations to produce a derivative protein, and the derivative protein has the same function as the protein in (a). 2. The use of a protein according to claim 1 as a tumor-testis antigen. 3. An application of the tumor antigen protein as claimed in claim 1 in the preparation of a drug for treating liver cancer. 4. The use of a tumor antigen protein as claimed in claim 1 in the preparation of a drug for treating intestinal cancer. 5. A DNA sequence encoding the tumor antigen protein of claim 1. 6. A DNA sequence according to claim 5, characterized in that it has the nucleotide sequence shown in Sequence 2, and its accession number in the gene library is: FATE AF249872. 7. The application of the gene as claimed in claim 5 as tumor-testis antigen gene. 8. A tumor antigen peptide having the same function as the peptide fragment produced by the tumor antigen protein according to claim 1, which has the amino acid sequence of 140th to 148th in the amino acid sequence of sequence 1. 9. The use of a tumor antigen peptide as claimed in claim 8 in the preparation of a drug for treating liver cancer. 10. The use of a tumor antigen peptide as claimed in claim 8 in the preparation of a drug for treating intestinal cancer.

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