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Genetically engineered antibody of anti-rabies virus as well as preparation method and application of genetically engineered antibody

A technology of genetic engineering antibody, rabies virus, applied in the field of genetic engineering

Active Publication Date: 2015-05-20
CHANGCHUN BCHT BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no genetically engineered antibody on the market at present, so research on recombinant engineered antibodies is still needed to obtain recombinantly engineered antibodies with high neutralizing activity against rabies virus

Method used

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  • Genetically engineered antibody of anti-rabies virus as well as preparation method and application of genetically engineered antibody
  • Genetically engineered antibody of anti-rabies virus as well as preparation method and application of genetically engineered antibody
  • Genetically engineered antibody of anti-rabies virus as well as preparation method and application of genetically engineered antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0168] Example 1: Acquisition of the gene encoding the recombinant anti-rabies virus antibody zipFv

[0169] 1. Codon optimization of zipFv57 gene

[0170] According to FV57 described in "Negative effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57" published by the inventor on Molecular Immunology VL85S Antibody (marked as scFv57 in the present invention) sequence, obtain the amino acid sequence of the light chain variable region (marked as 57VL) of the anti-rabies virus antibody and the heavy chain variable region (57VH) of the anti-rabies virus antibody. The heavy chain variable region of the obtained anti-rabies virus antibody, namely 57HL, has the amino acid sequence shown in SEQ ID NO:1; the light chain variable region of the obtained anti-rabies virus antibody, namely 57VL, has the amino acid sequence shown in SEQ ID NO:2 Amino acid sequence shown. Select codons favored by Escherichia coli to optimize the nucleic ...

Embodiment 2

[0187] Example 2: Expression, refolding assembly and purification of recombinant anti-rabies virus antibody zipFv

[0188] 1. Contains 57V H -Construction of the expression vector of the FOS-HA gene:

[0189] The 57V prepared by the correct embodiment 1 of the sequencing H -FOS-HA gene was digested with NdeI and XhoI, and the expression vector pET20b (purchased from Shanghai Tiancheng Technology Co., Ltd.) through the same double digestion, such as figure 2 Swimming lane 2, ligated with T4 ligase, then transformed into E.coli top10, small plasmid was extracted, and the clones with correct enzyme digestion results were sent for sequencing, and the clones consistent with the target sequence were selected, and the corresponding recombinant expression plasmid was marked as pET20b-57V H -FOS-HA.

[0190] 2. Contains 57V L -Construction of the expression vector of the JUN-His gene:

[0191] The 57V prepared by the correct embodiment 1 of the sequencing L - After the JUN-His g...

Embodiment 3

[0204] Example 3: Detection of specific antigen-binding activity of recombinant anti-rabies virus antibody zipFv57

[0205] The binding activity of zipFv57 compared to scFv57 was detected by Elisa method.

[0206] Rabies virus (aG strain, gifted by Jilin Maifeng Biological Pharmaceutical Co., Ltd.) was coated on a 96-well plate, and 100 μL was added to each well, scFv57 (preserved in this laboratory, and its preparation method was specifically shown in "Negative Immunology" published by the inventor on Molecular Immunology). Effects of a disulfide bond mismatch in anti-rabies G protein single-chain antibody variable fragment FV57》中FV57 VL85S Antibody preparation method) was used as a positive control, and an irrelevant protein with a histidine tag was used as a negative control, and the concentration of the protein used in each group was the same. Block with 3% BSA-PBS and incubate at 37°C for 1h. Add the antibody zipFv57 prepared in Example 2 diluted with PBS, 100 μL per we...

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Abstract

The invention belongs to the technical field of genetic engineering, and discloses a genetically engineered antibody of anti-rabies virus as well as a preparation method and an application of the genetically engineered antibody. The genetically engineered antibody provided by the invention is formed by assembling a heavy-chain variable area of a recombinant anti-rabies virus antibody and a light-chain variable area of the recombinant anti-rabies virus antibody; a leucine zipper is fused in the heavy-chain variable area of the recombinant anti-rabies virus antibody; and a leucine zipper is fused in the light-chain variable area of the recombinant anti-rabies virus antibody. The genetically engineered antibody is high in rabies virus neutralizing capability, is simple in structure and easy to built; high-efficient expression in a prokaryotic system can be realized, and the industrial mass production can be relatively easy to realize.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an anti-rabies virus genetic engineering antibody, its preparation method and application. Background technique [0002] Rabies is a 100% fatal infectious disease transmitted by animals. The disease is prevalent in more than 100 countries and regions. Globally, as many as 55,000 people die from rabies every year, and the number of rabies cases in my country is about 2,000. When severely bitten or scratched by animals carrying rabies virus, that is, rabies virus level III exposure, the World Health Organization recommends post-exposure prophylaxis (PEP) to prevent and treat rabies. Methods of post-exposure prophylaxis include wound washing, vaccination, and antibody infiltration into the wound. [0003] With the development of genetic engineering technology, injection of anti-rabies virus antibody has gradually become an important method for neutralizing r...

Claims

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Application Information

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IPC IPC(8): C07K16/10C07K19/00C12N15/13C12N15/62C12N15/70A61K39/205A61P31/14G01N33/569
Inventor 孔维吴永革李状谷铁军程月陈晓旭席华龙
Owner CHANGCHUN BCHT BIOTECH
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