CN1663603A - Application of a tumor-placenta antigen protein and its DNA in tumor therapy - Google Patents

Abstract

The invention relates to the use of tumor-placenta antigen protein in preparing medicament for treating cancer, the invention also relates to the use of the DNA sequence encoding the tumor-placenta antigen protein in preparing medicament for treating cancer, the tumor antigen protein can provide a new way for tumor treatment and the diagnosis of tumor occurrence, development and transfer.

Description

Application of a tumor-placenta antigen protein and its DNA in tumor therapy

technical field

The present invention relates to a tumor antigen protein and the application of its coding DNA, in particular to the application of a tumor-placenta antigen protein and its DNA in the preparation of medicines for treating liver cancer, intestinal cancer, gastric cancer and lung 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 the key to finding a new tumor treatment method A 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. At the same time, there are also some tumor antigen proteins that are not expressed in any normal tissue except the placenta, even in the testis. The placenta is a tissue that only pregnant women have. From the perspective of tumor immunotherapy application, this type of antigen is not expressed in the human body. It has similar properties to tumor-testis antigens, so it can also be classified as tumor-testis antigens or directly called tumor-placenta antigens.

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, intestinal cancer, gastric cancer and lung cancer are the most common malignant tumors in human tumors, especially in recent years, the incidence rate has gradually increased. These tumors grow rapidly and are difficult to cure even if they are found. At present, the main treatment methods are local treatments such as traditional surgical resection and radiotherapy. However, most patients are difficult to control when they are diagnosed, and they are quite resistant to radiotherapy and chemotherapy. . Therefore, the effect and prognosis of treatment are often poor, and the one-year and five-year survival rates of patients are very low. 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 tumors 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 the application of a tumor antigen protein in the preparation of medicine for treating cancer.

Another object of the present invention is to provide an application of a DNA sequence encoding a tumor antigen protein in the preparation of a drug for treating cancer.

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

The present invention provides an application of a tumor antigen protein in the preparation of a drug for treating cancer, the protein has the following amino acid sequence (a) or (b):

(a) have the amino acid sequence shown in Figure 7;

(b) A derivative protein produced by substituting, deleting or adding mutations to the amino acid sequence in (a), and the derivative protein has the same function as the protein in (a).

The tumor antigen protein of the present invention can produce peptide fragments that combine with major histocompatibility complex (MHC)-class I molecules and can be recognized by T cells in the combined state through intracellular decomposition to stimulate immune responses for the purpose of treating cancer.

When the tumor antigen protein of the present invention is used to prepare a drug for treating cancer, it is preferably used for treating liver cancer, intestinal cancer, gastric cancer or lung cancer. The tumor antigen protein of the present invention can also be used as a tumor-placental antigen to make a vaccine.

In another aspect, the present invention also provides an application of a DNA sequence encoding a tumor antigen protein in the preparation of a drug for treating cancer, wherein the encoded tumor antigen protein has the following amino acids as described in (a) or (b) sequence:

(a) having the amino acid sequence shown in Sequence 1;

(b) A derivative protein produced by substituting, deleting or adding mutations to the amino acid sequence in (a), and the derivative protein has the same function as the protein in (a).

Preferably, the accession number of the DNA in the gene library is: CP1, BC022335, such as the nucleotide sequence shown in SEQ ID No: 1 in the sequence listing.

Preferably, said DNA sequence is used for the treatment of liver cancer, intestinal cancer, gastric cancer or lung cancer. The DNA of the present invention can also be used as a tumor-placental gene for preparing vaccines.

The advantage of the present invention is that: the present invention identifies the CP1 gene as a tumor-placental antigen gene for the first time; when immunotherapy is used to treat cancer, the tumor antigen protein of the present invention can be used to induce the body to produce an immune response that only targets tumor cells , so that while not killing normal cells, it specifically kills tumor cells in the body to achieve the effect of treating or preventing tumor cell metastasis; at the same time, through the detection of the tumor antigen gene or antibody, the occurrence, development, and presence or absence of tumors can be detected. Transfer for diagnosis.

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

Description of drawings

Fig. 1 is the RT-PCR detection result of the gene PLAC1 encoding the antigenic protein of the present invention in 16 kinds of adult normal tissues;

Lanes 1 to 16 respectively represent adult normal tissues, spleen, prostate, ovary, small intestine, placenta, large intestine, white blood cells, testis, heart, lung, liver, brain, kidney, skeletal muscle, pancreas and thymus.

Fig. 2 is the RT-PCR result of the gene PLAC1 encoding the antigenic protein of the present invention in the cancer tissue of liver cancer and the paired paracancerous tissue

Among them, Ca represents the liver cancer tissue, and Adj represents the paired paracancerous tissue.

Fig. 3 is the Northern blot result of the gene PLAC1 encoding the antigenic protein of the present invention in liver cancer tissue and paired paracancerous tissue

Among them, Ca represents the liver cancer tissue, and Adj represents the paired paracancerous tissue.

Figure 4 is the expression result of CP1 gene recombinant protein in Escherichia coli

Among them, N represents no induction; 1 and 2 represent the whole protein of Escherichia coli that induces the expression of recombinant protein, compared with N, a protein band appears at the position of 23kD; 3 and 4 represent the expression of Escherichia coli that induces the expression of recombinant protein and is washed with inclusion bodies 5 and 6 represent the results of identifying the recombinant protein by using the monoclonal antibody against the 6 histidines contained in the recombinant protein by Western hybridization; the arrow shows the expression in Escherichia coli CP1 gene recombinant protein.

Figure 5 is the result of the polyclonal antibody produced by the antigen protein of the present invention detected by ELISA method to immunize rabbits

分别代表免疫三只不同的家兔产生的三份不同的血清；

代表作为阴性对照的没有用本发明抗原蛋白质免疫的家兔的血清。in

Respectively represent three different sera produced by immunizing three different rabbits;

It represents the serum of rabbits not immunized with the antigenic protein of the present invention as a negative control.

Figure 6 is the result of the expression of the antigenic protein of the present invention in gastric cancer tissue detected by immunohistochemistry

The arrows indicate the detected antigenic proteins of the present invention.

Figure 7: Amino acid sequence of the tumor antigen protein of the present invention.

Detailed ways

Example 1. Discovery of Differentially Expressed cDNA Fragment PLAC1 in Liver Cancer

In order to screen differentially expressed genes in primary hepatocellular carcinoma, we used suppression subtractive hybridization (suppression subtractive hybridization, SSH) kit (PCR-SelectTM cDNA Subtraction Kit) from Clontech Company, and used suppression subtractive hybridization technology to obtain liver cancer tissue As a tester (tester), the corresponding paracancerous tissue as a driver (driver), carried out suppression subtractive hybridization.

Firstly, the total RNA of liver cancer and adjacent tissues was extracted with TRIzol kit (Gibco Company), mRNA was isolated with PolyATract mRNA isolation kit of Promega Company (Promega, Madison, WI), and the concentration and purity of mRNA were detected by ultraviolet spectrophotometry.

The SSH operation process is according to the kit instructions. Synthesize the first and second strands of cDNA with 2 μg mRNA, then digest the double-stranded cDNA with RsaI enzyme, and extract and purify the cDNA fragments with phenol/chloroform. (adaptor) was ligated overnight at 16°C, and 1 μl of the ligated product was diluted in 200 μl of water to detect the ligation efficiency. Denature 1.5 μl of tester double-stranded DNA with different adapters, 1.5 μl driver double-stranded DNA and 1 μl 4× hybridization solution at 98°C for 1.5 minutes, and hybridize at 68°C for 8 hours. Then mix the two hybridization samples and an excess of freshly denatured driver, and hybridize at 68°C for 16 hours. After hybridization, add 200 μl diluent and dilute it as a template for two rounds of PCR amplification (primer PCR primer 1: 5'CTA ATA CGACTC ACT ATA GGG C 3'; nested PCR primer 1: 5'TCG AGC GGC CGCCCG GGC AGG T 3'; nested PCR primer 2R: 5'AGC GTG GTC GCGGCC GAG GT 3'), the first PCR cycle parameters are: 75°C for 5 minutes, 30 cycles: 94°C for 30 seconds, 66°C for 30 seconds, 72°C 1.5 minutes. After the first-round PCR product was diluted 1:10, 1 μl was used as a template for nested PCR (nested PCR): 15 cycles: 94°C for 10 seconds, 68°C for 30 seconds, and 72°C for 1.5 minutes. 8 μl of each of the first and second PCR products were analyzed by 2% agarose gel electrophoresis. Use the G3PDH (glyceraldehyde 3-phosphate dehydrogenase) 5' and 3' primers provided in the kit to detect the subtractive hybridization efficiency according to the kit instructions.

The second-round PCR product was cloned into pGEM-T Easy vector according to conventional methods, and transformed into competent E.coli TOP10F' bacteria to form a subtractive library. Clones were randomly selected. Fragments with different sizes were sent to Shanghai Jikang Company for sequencing, and the results obtained were compared with the nucleic acid sequences in GenBank using Blast software. It turned out that one of the fragments was a part of a known placenta-specific gene PLAC1. Therefore, we found that PLAC1 was also expressed in liver cancer tissues.

Embodiment 2, using the method of RT-PCR to prove the expression of PLAC1 gene in liver cancer, intestinal cancer, gastric cancer and lung cancer

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 39 liver cancer patients, cDNAs from cancer tissues and matched paracancerous tissues of 24 intestinal cancer patients, cancer tissues and matched cancer tissues of 24 gastric cancer patients The cDNA of paracancerous tissue, the cDNA of cancer tissue and paired paracancerous tissue from 24 cases of lung cancer patients were used to detect the expression of PLAC1 gene in normal tissue and tumor tissue.

The conditions for RT-PCR in the present invention: use the advanced hot-start DNA polymerase of CLONTECH Company, 94°C for 15 seconds; 65°C for 30 seconds; 72°C for 40 seconds for 30 cycles, and then 72°C for 6 minutes. The primer sequence for RT-PCR, forward primer: ctg ttc ctg gcaccc tgt gca tcc; reverse primer: gat gcc gcc atg ctg ttc acc c. It was found that: PLAC1 gene was expressed in 50% of liver cancer specimens, 36% of intestinal cancer, 50% of gastric cancer and 40% of lung cancer, but not in the paired paracancerous tissues; in 16 important adult normal tissues , PLAC1 gene is only expressed in placental tissue (Figure 1, Figure 2). This example proves that the tumor-placental antigen gene PLAC1 is widely expressed in various tumor tissues. We renamed it as CP1 based on the comprehensive properties of the now known PLAC1 gene (the accession number in the gene library is: CP1, BC022335).

Example 3. Using Northern hybridization to analyze the expression of CP1 gene in tumor tissues of liver cancer, intestinal cancer, gastric cancer and lung cancer

Total RNA was extracted from cancerous tissues and paired paracancerous tissues of tumor patients with TRIZOL reagent (PROMEGA), and then mRNA was isolated from total RNA using mRNA isolation kit (PROMEGA). 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 tumor-placenta antigen protein gene CP1 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 results showed that the mRNA of the tumor antigen protein gene of the present invention was only expressed in the cancer tissues of liver cancer, intestinal cancer, gastric cancer and lung cancer, but not in the paired paracancerous tissues ( FIG. 3 ). It is consistent with the characteristics of tumor-placental antigen gene.

Embodiment 4, expression of CP1 gene recombinant protein in Escherichia coli

The pQE30 prokaryotic expression vector (purchased from INVOTRIGEN Company) was used to construct the plasmid pQE30-CP1 expressing tumor-placental antigen CP1. The plasmid was grown in Escherichia coli strain M15 (purchased from INVOTRIGEN Company) at 37°C, and the recombinant protein was induced by IPTG Express for 6 hours. 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 Escherichia coli using a nickel ion affinity chromatography column (purchased from INVOTRIGEN Company). The recombinant protein was identified by Western hybridization using a monoclonal antibody against the 6 histidines contained in the recombinant protein (purchased from INVOTRIGEN Company) ( FIG. 4 ). The results showed that there was an expected protein at the expected size of 23kD, and Western hybridization with anti-6 histidine monoclonal antibody confirmed that the expressed protein was the recombinant protein of CP1 gene.

Example 5. Detection of Anti-CP1 Recombinant Protein Antibodies in the Serum of Patients with Liver Cancer, Colon Cancer, Gastric Cancer and Lung Cancer by Western Hybridization

According to the method in Journal of Experimental Medicine, 187:1349, 1998, the serum of tumor patients with positive CP1 mRNA transcripts was screened, and it was found that there were antibodies against CP1 recombinant protein in the serum of patients with tumors (liver cancer, colon cancer, gastric cancer and lung cancer) . Therefore, CP1 is a new tumor-placental 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 6, Preparation of Artificial Anti-CP1 Recombinant Protein Polyclonal Antibody

Refer to the Molecular Cloning Experiment Guide, Second Edition (translated by Li Mengfeng and Jin Dongyan): 852, the method in the first and second sections of Chapter 18, immunize 3 rabbits 4 times with the purified CP1 recombinant protein, and immunize for the first time The second immunization was carried out one month after the second immunization, and the interval between the second and third immunization, the third and the fourth immunization was 2 weeks, and the serum of rabbits was harvested two weeks after the fourth immunization, anti-CP1 recombinant protein The polyclonal antibodies are in these sera. After diluting the polyclonal antibody at 1:500, serially dilute the polyclonal antibody by 3 times sequentially, and identify the polyclonal antibody by ELISA method. The results showed that the polyclonal antibodies produced by the three rabbits could all react specifically with the CP1 recombinant protein, and the titers of the polyclonal antibodies were very high ( FIG. 5 ).

Example 7. Detecting the Existence of Natural CP1 Antigen Protein in the Tumor Tissues of Patients with Liver Cancer, Colon Cancer, Gastric Cancer and Lung Cancer by Immunohistochemical Method

Referring to the method in LABORATORY INVESTIGATION, 83:1, 2003, the purified artificial anti-CP1 recombinant protein polyclonal antibody was used to perform immunohistochemical experiments to detect tumor tissues of patients with liver cancer, colon cancer, gastric cancer and lung cancer. Whether there is a natural CP1 antigen protein, it was found that there is a natural CP1 protein in these tumor tissues, and the protein is located in the nucleus. Figure 6 shows the expression results of CP1 antigen protein in gastric cancer. Therefore, CP1, as a new tumor-placental antigen, does exist in tumor tissue, which provides a further basis for the application of CP1 in clinical immunotherapy of tumors.

Example 8, Preparation of Mutant Tumor Antigen Protein and Screening of Serum Antibody

Referring to the Molecular Cloning Experiment Guide, Second Edition (translated by Li Mengfeng and Jin Dongyan): 693, the method in Chapter 15, the following random mutation DNA was prepared, the 361st base in sequence number 2 was mutated from A to G , thus the 24th amino acid in SEQ ID NO: 1 was mutated from S to G; the 661st base in SEQ ID NO: 2 was mutated from C to A, thus the 124th amino acid in SEQ ID NO: 1 was mutated Amino acids were mutated from L to I. The two mutated DNAs were respectively cloned into expression vectors to express the mutated tumor antigens. 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.

dongxueyuan-sequence list(1)

SEQUENCE LISTING

<110> Peking University

<120> Application of a tumor-placenta antigen protein and its DNA in tumor therapy

<130>PI000

<160>2

<170>Patent In version 3.1

<210>1

<211>1131

<212>DNA

<213>Homo sapiens

<220>

<221> CDS

<222>(292)..(930)

<223>

<400>1

ggcacgagga tcagaccatc agaaggattt gtataaagag tgactctcct atgaaggtaa 60

aggccacccc tcttcagttc cggtgactga gatacatttt tccaatcctg ggggcaaata 120

cagacacagc aagttccttc ttccctttgg aaatttggca gctgccttca ccagtgagca 180

caaagccaca tttcaaagga aactgacaaa ttatccccag ctgccagaag aagaaatcct 240

cactggacgg cttcctgttt cctgtggttc attatctgat tggctgcagg g atg aaa 297

Met Lys

1

gtt ttt aag ttc ata gga ctg atg atc ctc ctc acc tct gcg ttt tca 345

Val Phe Lys Phe Ile Gly Leu Met Ile Leu Leu Thr Ser Ala Phc Ser

5 10 15

gcc ggt tca gga caa agt cca atg act gtg ctg tgc tcc ata gac tgg 393

Ala Gly Ser Gly Gln Ser Pro Met Thr Val Leu Cys Ser Ile Asp Trp

20 25 30

ttc atg gtc aca gtg cac ccc ttc atg cta aac aac gat gtg tgt gta 441

Phe Met Val Thr Val His Pro Phe Met Leu Asn Asn Asp Val Cys Val

35 40 45 50

cac ttt cat gaa cta cac ttg ggc ctg ggt tgc ccc cca aac cat gtt 489

His Phe His Glu Leu His Leu Gly Leu Gly Cys Pro Pro Asn His Val

55 60 65

cag cca cac gcc tac cag ttc acc tac cgt gtt act gaa tgt ggc atc 537

Gln Pro His Ala Tyr Gln Phe Thr Tyr Arg Val Thr Glu Cys Gly Ile

70 75 80

agg gcc aaa get gtc tct cag gac atg gtt atc tac agc act gag ata 585

Arg Ala Lys Ala Val Ser Gln Asp Met Val Ile Tyr Ser Thr Glu Ile

85 90 95

cac tac tct tct aag ggc acg cca tct aag ttt gtg atc cca gtg tca 633

His Tyr Ser Ser Lys Gly Thr Pro Ser Lys Phe Val Ile Pro Val Ser

100 105 110

tgt gct gcc ccc caa aag tcc cca tgg ctc acc aag ccc tgc tcc atg 681

Cys Ala Ala Pro Gln Lys Ser Pro Trp Leu Thr Lys Pro Cys Ser Met

115 120 125 130

aga gta gcc agc aag agc agg gcc aca gcc cag aag gat gag aaa tgc 729

Arg Val Ala Ser Lys Ser Arg Ala Thr Ala Gln Lys Asp Glu Lys Cys

135 140 145

tac gag gtg ttc agc ttg tca cag tcc agt caa agg ccc aac tgc gat 777

Tyr Glu Val Phe Ser Leu Ser Gln Ser Ser Gln Arg Pro Asn Cys Asp

150 155 160

dongxueyuan-sequence list (1)

tgt cca cct tgt gtc ttc agt gaa gaa gag cat acc cag gtc cct tgt 825

Cys Pro Pro Cys Val Phe Ser Glu Glu Glu His Thr Gln Val Pro Cys

165 170 175

cac caa gca ggg gct cag gag gct caa cct ctg cag cca tct cac ttt 873

His Gln Ala Gly Ala Gln Glu Ala Gln Pro Leu Gln Pro Ser His Phe

180 185 190

ctt gat att tct gag gat tgg tct ctt cac aca gat gat atg att ggg 921

Leu Asp Ile Ser Glu Asp Trp Ser Leu His Thr Asp Asp Met Ile Gly

195 200 205 210

tcc atg tga tcctcaggtt tggggtctcc tgaagatgct atttctagaa 970

Ser Met

ttagtatata gtgtacaaat gtctgacaaa taagtgctct tgtgaccctc atgtgagcac 1030

ttttgagaaa gagaaaccta tagcaacttc atgaattaag cctttttcta tatttttata 1090

ttcatgtgta aacaaaaaat aaaataaaat tctgatcgca t 1131

<210>2

<211>212

<212>PRT

<213>Homo sapiens

<400>2

Met Lys Val Phe Lys Phe Ile Gly Leu Met Ile Leu Leu Thr Ser Ala

1 5 10 15

Phe Ser Ala Gly Ser Gly Gln Ser Pro Met Thr Val Leu Cys Ser Ile

20 25 30

Asp Trp Phe Met Val Thr Val His Pro Phe Met Leu Asn Asn Asp Val

35 40 45

Cys Val His Phe His Glu Leu His Leu Gly Leu Gly Cys Pro Pro Asn

50 55 60

His Val Gln Pro His Ala Tyr Gln Phe Thr Tyr Arg Val Thr Glu Cys

65 70 75 80

Gly Ile Arg Ala Lys Ala Val Ser Gln Asp Met Val Ile Tyr Ser Thr

85 90 95

Glu Ile His Tyr Ser Ser Lys Gly Thr Pro Ser Lys Phe Val Ile Pro

100 105 110

Val Ser Cys Ala Ala Pro Gln Lys Ser Pro Trp Leu Thr Lys Pro Cys

115 120 125

Ser Met Arg Val Ala Ser Lys Ser Arg Ala Thr Ala Gln Lys Asp Glu

130 135 140

Lys Cys Tyr Glu Val Phe Ser Leu Ser Gln Ser Ser Gln Arg Pro Asn

145 150 155 160

Cys Asp Cys Pro Pro Cys Val Phe Ser Glu Glu Glu His Thr Gln Val

165 170 175

Pro Cys His Gln Ala Gly Ala Gln Glu Ala Gln Pro Leu Gln Pro Ser

dongxueyuan-sequence list(1)

180 185 190

His Phe Leu Asp Ile Ser Glu Asp Trp Ser Leu His Thr Asp Asp Met

195 200 205

Ile Gly Ser Met

210

Claims (7)

1. The application of a tumor antigen protein in the preparation of a medicament for treating cancer, the protein has the amino acid sequence described in (a) or (b): (a) have the amino acid sequence shown in Figure 7; (b) A derivative protein produced by substituting, deleting or adding mutations to the amino acid sequence in (a), and the derivative protein has the same function as the protein in (a). 2. The application of the protein according to claim 1, characterized in that the cancer is liver cancer, intestinal cancer, gastric cancer or lung cancer. 3. The application of the protein according to claim 1, characterized in that the drug is a vaccine prepared from the protein as a tumor-placental antigen. 4. An application of a DNA sequence encoding a tumor antigen protein in the preparation of a medicament for treating cancer, wherein the encoded tumor antigen protein has the amino acid sequence described in (a) or (b): (a) having the amino acid sequence shown in Sequence 1; (b) A derivative protein produced by substituting, deleting or adding mutations to the amino acid sequence in (a), and the derivative protein has the same function as the protein in (a). 5. The application of DNA according to claim 4, characterized in that the DNA sequence is the accession number in the gene library: CP1, BC022335. 6. The application of DNA according to claim 4, characterized in that said cancer is liver cancer, intestinal cancer, gastric cancer or lung cancer. 7. The application of DNA according to claim 4, characterized in that said medicine is a vaccine made from said DNA as a tumor-placental gene.

Images