Antigenic determinant polypeptide of human tumor-testis antigen HCA587 and use thereof

An antigenic determinant, testis antigen technology, applied in the direction of antineoplastic drugs, animal/human peptides, medical preparations containing active ingredients, etc.

Inactive Publication Date: 2005-06-01
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the 12 patients treated, 7 were NY-ESO-1 serum antibody negative and 5 were positive; after the former was immunized with the vaccine, 4/7 patients developed NY-ESO-1 peptide

Method used

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  • Antigenic determinant polypeptide of human tumor-testis antigen HCA587 and use thereof
  • Antigenic determinant polypeptide of human tumor-testis antigen HCA587 and use thereof
  • Antigenic determinant polypeptide of human tumor-testis antigen HCA587 and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. Gene discovery process of CT antigen HCA587

[0027] In order to screen abnormally expressed CT antigens in primary hepatocellular carcinoma, we used the cDNA synthesis Kit, Uni-ZAP XR vector and Gigapack IIIGOLD packaging extract kit from Stratagene to select tissue markers from patients with hepatocellular carcinoma for SEREX analysis. filter.

[0028] First, extract total RNA from liver cancer and paracancerous tissues according to the operating instructions of the TRIzol kit (Gibco Company). The steps include: tissue homogenate, extraction, RNA precipitation, washing and dissolving. Ultrapure water dissolves the precipitate. The OD260 and OD280 values ​​of the samples were measured by ultraviolet spectrophotometer, and the RNA quality (the ratio of 28S and 18S rRNA) was detected by formaldehyde denaturing gel electrophoresis.

[0029] The PolyATract mRNA Isolation Kit (Promega, Madison, WI) from Promega Company was used to isolate mRNA. oligo(dT) bindi...

Embodiment 2

[0036] Example 2. Expression of HCA587 recombinant protein and its distribution in tumor tissue

[0037] (1) Expression of recombinant pQE30-HCA587 protein

[0038] Design specific primers according to the nucleotide sequence of HCA587, synthesize BamHI and HindIII restriction sites at the 5' ends of the upstream and downstream primers, P1: 5'ATC GGATCC CCTCCCGTTCCAGGCGT3',P2:5'ACT AAGCTT TCACTCAGAAAAGGAGAC3', using the cDNA of normal testis tissue as a template, the full-length ORF of HCA587 was amplified by PCR, and after the PCR product was connected with the pGEM-Teasy vector, it was transformed into DH5a, and the positive clones containing the correct insertion of HCA587 were selected, and double-expressed with BamHI and HindIII, respectively. Digest the HCA587-pGEMT-easy plasmid and the pQE-30 vector, recover the HCA587 and pQE-30 fragments from the gel after electrophoresis, connect with T4 ligase overnight, transform XL-Blue competent cells, and extract the recombina...

Embodiment 3

[0060] Example 3. T2 binding assay

[0061]To analyze HCA587 to induce the body to produce an immune response, it is first required that the protein is processed by APC cells, and the corresponding polypeptide is combined with its HLA molecule and presented to the cell surface. Therefore, we first used the model invented by Dr.Kenneth Parker (Parker, K.C., et al.1994.J.Immunol.152:163) to predict the polypeptide that can bind to HLA-A2 in the HCA587 protein sequence (web site: http: / / www-bimas.cit.nih.gov / molbio / hla_bind / ), combined with other HLA-A2 predicted binding peptide software, screened out 9 more likely antigenic peptides bound to HCA587 and HLA-A2, and synthesized all of them with a peptide synthesizer in vitro The predicted polypeptide (see Table 2) was purified by HPLC with a purity of 99%, and then the cell line CEMX721.174.T2 (T2 for short) was used for HLA-A2 binding test. Because T2 cells only express HLA-A2 molecules, but cannot present endogenous polypeptid...

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Abstract

The present invention discloses antigenic determinant polypeptide on human tumor-testis antigen HCA587. The polypeptide and its derivative can combine with human HLA molecule and lead target cell to crack via cytotoxic T lymphocyte. The present invention also discloses the application of these antigen polypeptides and their derivatives in the diagnosis, prevention and treatment of liver cancer and other tumors.

Description

technical field [0001] The present invention relates to a class of antigenic determinant polypeptides, especially the antigenic determinant polypeptides on human tumor-testis antigen HCA587, and further relates to the application of these antigenic peptides and their derivatives in the treatment, diagnosis and prevention of liver cancer and other tumors . Background technique [0002] The occurrence of tumor is a multi-stage and multi-step process, which is accompanied by many cytological and genetic changes in the body, and the abnormal expression of various genes, including the activation of oncogenes and / or the inactivation of tumor suppressor genes , to stimulate abnormal signal transduction pathways, resulting in abnormal cell cycle and apoptosis, resulting in malignant transformation of cells, and with the accumulation of various molecular abnormalities, tumor progression and metastasis occur. Therefore, understanding these genes specifically expressed in tumor tissue...

Claims

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Application Information

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IPC IPC(8): A61K38/18A61K39/00A61K48/00A61P35/00C07H21/04C07K14/435
Inventor 陈慰峰李兵王月丹
Owner PEKING UNIV
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