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Recombined composite epitope antigen of liver cancer, preparation method, and application of preparing medication for specific immunity treatment of liver cancer

A technology of compound epitopes and antigens, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., to achieve the effect of increasing MHC coverage, reducing immune evasion and broad application prospects

Inactive Publication Date: 2007-05-09
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application of simple peptide vaccines has its limitations, such as only being used in conjunction with specific HLA types

Method used

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  • Recombined composite epitope antigen of liver cancer, preparation method, and application of preparing medication for specific immunity treatment of liver cancer
  • Recombined composite epitope antigen of liver cancer, preparation method, and application of preparing medication for specific immunity treatment of liver cancer
  • Recombined composite epitope antigen of liver cancer, preparation method, and application of preparing medication for specific immunity treatment of liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Molecular Design and Gene Synthesis of Liver Cancer Compound Epitope Antigen

[0031] 1. Materials:

[0032] Prokaryotic expression vector pET22b is a product of Novagen; prokaryotic expression vector pBVIL1: has applied for a Chinese patent and obtained authorization, patent number: ZL00100695.9, patent name: "expression vector pBVIL1 and its construction method and use", and has been preserved, preserved No.: CGMCC No: 0437; host bacteria Escherichia coli JM109 and HB101 are commercially available and kept in our laboratory; high-fidelity Pyrobest TM DNA polymerase, DNA restriction endonucleases Xho I, Xba I, Nde I and HindIII, DNA relative molecular quality standards DL2000, DL 15000 were purchased from Dalian Bao Biological Company; cloning vector pGEM-T Easy, DNA restriction endonucleases EcoR I, BamHI, and T4 DNA ligase are products of Promega Company; common Taq DNA polymerase is a product of BBI Company; PCR product purification kit and plasmid extrac...

Embodiment 2

[0064] Example 2 Expression and Purification of Liver Cancer Compound Epitope Antigen

[0065] 1. Materials:

[0066] SDS-PAGE low molecular weight standard protein is the product of Shanghai Institute of Biochemistry; chromatography media SephadexG-50, Q-Sepharose Fast Flow, S-Sepharose Fast Flow are products of Pharmacia; Kodak automatic gel imaging system; BIO-RAD protein electrophoresis instrument. Yu ditto.

[0067] 2. Method results:

[0068] 1. Expression and purification of antigen

[0069] 1.1 Cultivation of expression strains:

[0070] Pick a single clone and shake it overnight at 37°C, and inoculate it in LB (Amp + ) culture medium, activate at 37°C until the A value reaches 0.4-0.6, continue culturing in a water-bath shaker at 42°C for 5 hours, and collect the bacteria. Take 1ml of induced bacterial pellet, resuspend in 1×SDS loading buffer, boil at 100°C for 5min, centrifuge at 1000r / min for 3min, take 10μl of lysate for 15% SDS-PAGE electrophoresis, stain wi...

Embodiment 3

[0076] Example 3 In Vitro Transduction Activity Detection of Liver Cancer Compound Epitope Antigen

[0077] 1. Materials:

[0078] Tat+T, T purified protein prepared in Example 2; golden hamster kidney BHK cells and mouse myeloma cells SP2 / 0 are commercially available and stored in our laboratory; RPMI 1640 medium and DMEM medium are products of GibcoBRL; refined fetal Bovine serum was purchased from Jianghai Biological High-tech Development Co., Ltd., Tongjiang City, Heilongjiang; BIO-RAD protein electrophoresis instrument; HRP-labeled goat anti-mouse IgG and FITC-labeled goat anti-mouse IgG were purchased from Zhongshan Company; anti-His monoclonal antibody was The product of Invitrogen Company; the PVDF membrane used for Western blotting was the product of Pharmacia Company; the ECL system was purchased from Pierce Company. Yu ditto.

[0079] 2. Method results:

[0080] Add the Tat+T fusion protein to a 6-well plate containing BHK and SP2 / 0 cells, incubate at 37°C for 2 ...

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Abstract

This invention discloses recombinant hepatocarcinoma composite epitope antigen, and its preparation method and application in manufacturing hepatocarcinoma-specific immunotherapy drugs. The composite epitope antigen has transduction activity, and contains T cell epitopes of several tumor antigens. The composite epitope antigen is obtained by gene synthesis and molecular cloning, and has such advantages as high safety and stability, and reduced immune evasion. The composite epitope antigen has potential applications in specific immunotherapy of liver cancer.

Description

technical field [0001] The invention relates to a recombinant antigen in the field of bioengineering, in particular to a recombinant liver cancer compound epitope antigen, and also relates to a preparation method of the antigen and its application in preparing liver cancer-specific immunotherapy drugs. Background technique [0002] Primary liver cancer (HCC) is one of the most common malignant tumors in the world. my country is one of the countries with the highest incidence of liver cancer in the world. The patients with liver cancer in my country account for about 50% of the world, and the 5-year survival rate of patients is less than 12%. %, the mortality rate is as high as 20 / 10 per year 5 . Therefore, it is of great significance to strengthen the research on the prevention and treatment of liver cancer in our country. [0003] At present, the main treatment methods for liver cancer are traditional surgical resection, local or systemic chemotherapy, etc. Although the lo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K14/00C12N15/12C12N15/11C12N15/70A61K38/17A61P35/00A61P37/02
Inventor 何竞凌世淦张贺秋宋晓国王国华
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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