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Multi sub-gene resistant type HCV (Hepatitis C Virus) antibody gene R3-19 and application thereof

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

Inactive Publication Date: 2016-01-06
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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  • Multi sub-gene resistant type HCV (Hepatitis C Virus) antibody gene R3-19 and application thereof
  • Multi sub-gene resistant type HCV (Hepatitis C Virus) antibody gene R3-19 and application thereof
  • Multi sub-gene resistant type HCV (Hepatitis C Virus) antibody gene R3-19 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, primer design

[0023] Design human IgG, IgM heavy chain constant region and κ, λ light chain constant region mRNA gene-specific reverse transcription primers according to John McCafferty's report, as well as PCR amplification primers for heavy chain and light chain variable region (McCaffertyJ, GriffithsAdFau-WinterG, WinterGFau - Chiswell DJ, Chiswell DJ. Phage antibodies: filamentous phage displaying antibody variable domains. Nature. 1990; 348(6301):552-4.). To facilitate insertion into the T7 phage vector, EcoRI and HindIII 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 reverse primer and the 5' end of the VH ...

Embodiment 2

[0028] Example 2, mRNA extraction and cDNA synthesis

[0029] Combining RNA / DNAStabilizationReagentforBlood / BoneMarrow(Roche) and mRNAisolationkitforblood / bonemarrow(Roche), use the magnetic bead method to directly isolate and extract mRNA from peripheral blood leukocytes, operate according to the instructions, and measure the extracted mRNA concentration and A260 / A280 value. The results showed that an average of 130 ng mRNA was obtained from anticoagulated peripheral blood cells per milliliter 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, cDNA was synthesized by IIFirstStrandcDNASynthesisKit (NEB), combined, and stored at -20°C for future use...

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 50 μL of PCR volume, each contains 25 pmoL of single upstream primer and premixed reverse primer, 2 μL of cDNA, 10 μL of 5×ReactionBuffer, 10 μL of 5×HighGCEnhancer, 1 μL of 10mmoldNTP, 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. PCR products were subjected to agarose gel electrophoresis, and HighPurePCRProductPurificationKit (Roche) was used to purify PCR. The electrophoresis re...

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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 (hepatitsCvirus, HCV) infected people 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 obvious ...

Claims

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Application Information

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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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