Anti-multi-subgenotype HCV antibody gene r3-19 and its application

A genotype and antibody technology, applied in the field of biochemistry, can solve the problems of differences in the neutralization ability of different genotypes, and achieve the effect of extensive binding characteristics and neutralization ability

Inactive Publication Date: 2020-06-02
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, not all reported broadly cross-binding antibodies have neutralizing effects on all genotypes of HCV, and there are differences in the neutralizing ability for different genotypes

Method used

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  • Anti-multi-subgenotype HCV antibody gene r3-19 and its application
  • Anti-multi-subgenotype HCV antibody gene r3-19 and its application
  • Anti-multi-subgenotype HCV antibody gene r3-19 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, primer design

[0023] According to the report of John McCafferty, the mRNA gene-specific reverse transcription primers for human IgG, IgM heavy chain constant region and κ, λ light chain constant region, as well as PCR amplification primers for heavy chain and light chain variable region were designed (McCafferty J, Griffiths AdFau- Winter G, Winter G Fau-Chiswell DJ, Chiswell DJ. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990; 348(6301): 552-4.). In order to facilitate insertion into the T7 phage vector, EcoR I and Hind III restriction sites were added to the 5' end of the forward primer for the variable region of the light chain (VL) and the 5' end of the reverse primer for the variable region of the heavy chain (VH). In order to generate the VL-linker-VH gene fragment, the PCR-amplified VL and VH fragments were connected by overlapping extension PCR, and the reverse overlaps were added to the 5' end of the VL rev...

Embodiment 2

[0028] Example 2, mRNA extraction and cDNA synthesis

[0029] Combining RNA / DNA Stabilization Reagent for Blood / Bone Marrow (Roche) and mRNA isolation kit for blood / bone marrow (Roche), using the magnetic bead method to directly isolate and extract mRNA from peripheral blood leukocytes, operate according to the instructions, and measure the concentration of extracted mRNA and A260 / A280 values. The results showed that an average of 130 ng of mRNA was obtained per milliliter of anticoagulated peripheral blood cells of the patient, and the A260 / A280 value was 1.90-2.00, indicating that the isolated and extracted mRNA can be used for library construction, and then stored at -80°C for later use. Take 50ng of mRNA extracted from all samples as a template, respectively use human IgG, IgM heavy chain constant region, κ, λ light chain constant region mRNA gene-specific reverse transcription primers, II FirstStrand cDNA Synthesis Kit (NEB) synthesized cDNA, combined cDNA, and stored a...

Embodiment 3

[0030] Embodiment 3, single-chain variable region fragment (scFv) amplification

[0031] The heavy chain and light chain variable region genes were amplified by PCR, and the specific operation method was as follows: the heavy chain and the reverse primers for the variable region of the κ and λ light chains were premixed at equimolar concentrations, and the final concentration was 10 μM. In 50μL PCR volume, each contains 25pmoL of single upstream primer and premixed reverse primer, 2μL of cDNA, 10μL of 5×Reaction Buffer, 10μL of 5×High GC Enhancer, 1μL of 10mmol dNTP, High-Fidelity DNA Polymerase (NEB) 1 unit. Reaction conditions: 98°C pre-denaturation for 30 seconds, 98°C denaturation for 10 seconds, 60°C annealing for 30 seconds, 72°C extension for 30 seconds, a total of 25 cycles; finally 72°C extension for 2 minutes. The PCR product was subjected to agarose gel electrophoresis, and was purified by High Pure PCR Product Purification Kit (Roche). The electrophoresis results...

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Abstract

The invention discloses a multi sub-gene resistant type HCV (Hepatitis C Virus) antibody gene R3-19, wherein the heavy chain amino acid sequence thereof is shown by SEQ ID NO.38, and the light chain amino acid sequence is shown by SEQ ID NO.39, the antibody gene is obtained by vanning in an independently-constructed anti-HCV scFv antibody library through E2 region aa412-423 linear epitope (an HCV CD81 receptor binding epitope) synthetic peptide which is highly conservative in various subtype HCVs; the antibody library is formed by 39 parts of anti-HCV antibody positive peripheral blood samples of Chinese patients, the HCVs infected by the patients cover the main HCV popular subtypes in China, therefore, the R3-19 is an anti-HCV antibody which is typical in China and has wide binding characteristic, and has excellent application value in the fields of HCV diagnosis, HCV treatment, vaccine research and development and the like in China.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to the anti-multi-subgenotype HCV antibody gene R3-19, and also relates to the application of the antibody gene. Background technique [0002] So far, there are about 170 million hepatitis C virus (hepatits C virus, HCV) infected persons in the world, accounting for 3% of the world's total population. WHO estimates that new cases of HCV infection worldwide are increasing at a rate of 3 to 4 million per year. HCV is mainly transmitted through blood, and the proportion of chronicity after infection is as high as 80%, which is an important cause of liver cirrhosis and liver cancer. HCV belongs to the genus Hepatitis C virus of the Flaviviridae family and is a single-stranded positive-sense RNA virus. The HCV genome is highly variable, and according to the heterogeneity of the genome, it is divided into 7 genotypes and nearly 100 subtypes. In the world, HCV infection has obvio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/13C07K16/10C12N7/01G01N33/569A61K39/42A61P31/14
Inventor 兰林崔传佳朱研胡亚君毛青
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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