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Gene cloning of polyepitope antigen of hepatitis C virus and its coding sequence

A hepatitis C virus and coding sequence technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, gene therapy, antiviral agent, etc., can solve the problem that the epitope is not ideal and cannot be used Hepatitis C vaccine preparation, unsatisfactory effect and other issues

Inactive Publication Date: 2006-09-06
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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Problems solved by technology

[0006] However, so far, reports on the synergistic effect between B cell epitopes, CTL epitopes, and Th epitopes cannot be used for hepatitis C vaccine preparation and other purposes because the selected epitopes are not ideal and the effect is not ideal.

Method used

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  • Gene cloning of polyepitope antigen of hepatitis C virus and its coding sequence
  • Gene cloning of polyepitope antigen of hepatitis C virus and its coding sequence
  • Gene cloning of polyepitope antigen of hepatitis C virus and its coding sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0027] The specific construction method of hepatitis C virus multi-epitope antigen gene clone (hcvme) is as follows:

[0028] 1. Synthesizing 26 oligonucleotide fragments required for the hcvme gene (see SEQ ID NO.2, synthesized by Shanghai Shenergy Gambling Biotechnology Co., Ltd.).

[0029] 2. The 26 oligonucleotide fragments are both primers and templates. The two gene fragments A and B before and after the PCR reaction are respectively synthesized. Both PCR synthesis and splicing reactions use Expand TM Long Template PCR System.

[0030] 1. The synthesis of gene fragment A in the front of hcvme ( figure 2 )

[0031] 0.5 μl each of oligonucleotide fragments 1-14 (30 μM)

[0032] dNTP (10mM) 2.0μl

[0033] 10×PCR buffer 5.0μl

[0034] DNA polymerase mixture (10 units / μl) 1.0μl

[0035] Sterilized redistilled water 35μl

[0036] The PCR reaction was performed under the following conditions: 35 cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 74°C for 1 minute,...

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Abstract

The present invention relates to biomedicine engineering technology, and is the gene cloning of polyepitope antigen of hepatitis C virus hcvme and the coded polyepitope antigen HCVME. The hcvme includes 9 epitope genes of HVR1 simulating B cell and 4 epitope genes of T cell in E2 region of HCV genome, and the 4 epitope genes of T cell includes 2 of conservative CTL epitope in region C, 1 of conservative CTL epitope in region NS3 and 1 of conservative Th epitope in region NS3. Serial tests show that hcvme has high cross reaction with karyogamy expressed product GST-HCVME of gst gene and positive hepatitis C antibody serum, and the GST-HCVME immunized male rabbit serum has high distinction frequency on natural HVR1 synthetic peptide. Therefore, hcvme and its expressing product HCVME may be used in preparing nucleic acid vaccine and polypeptide vaccine for hepatitis C and the test reagent for HCV antigen and antibody.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, and relates to the application of gene clone hcvme of hepatitis C virus multi-epitope antigen and its coding sequence HCVME in the preparation of hepatitis C vaccine. Background technique [0002] Hepatitis C virus (HCV) used to be known as non-A non-B hepatitis (NANBH) virus transmitted outside the intestines (through blood), and it was officially named after the gene sequence of about 75% of its 3' end was determined in 1989. It has the characteristics of worldwide distribution, wide transmission route, easy mutation of genome, high rate of slowing down, mixed infection with other types of hepatitis, and causing liver cirrhosis and hepatocellular carcinoma. HCV belongs to the family Flaviviridae. The viral genome is a linear single-stranded RNA with a length of about 9500 nucleotides. ~5) Composition. The HCV genome is highly variable and often exists...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/13C12N15/51C07K16/28A61K48/00A61K39/29G01N33/576A61P31/12
Inventor 戚中田高军赵平龚育平赵兰娟潘欣
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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