Preparation method of pig-source single-chain genetic engineering antibody against foot-and-mouth disease viruses

A genetically engineered antibody, anti-foot-and-mouth disease virus technology, applied in genetic engineering, virus/phage, antiviral immunoglobulin, etc., can solve problems such as differences in B cell diversity

Inactive Publication Date: 2019-10-25
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since pigs are the natural host of FMDV infection, and there is a large difference in the diversity of B cells between mice and pigs, FMDV-specific porc...

Method used

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  • Preparation method of pig-source single-chain genetic engineering antibody against foot-and-mouth disease viruses
  • Preparation method of pig-source single-chain genetic engineering antibody against foot-and-mouth disease viruses
  • Preparation method of pig-source single-chain genetic engineering antibody against foot-and-mouth disease viruses

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preparation example Construction

[0037] The invention provides a kind of preparation method of anti-foot-and-mouth disease virus pig source single-chain genetic engineering antibody The preparation method of antibody comprises the following steps:

[0038] 1) separating peripheral blood mononuclear cells from pig blood samples immunized with foot-and-mouth disease virus antigen;

[0039] 2) FMDV 146S antigen is subjected to ultrafiltration and centrifugation to obtain concentrated FMDV 146S antigen, dyeing is carried out to the concentrated FMDV 146S antigen with dye, and the stained antigen is subjected to ultrafiltration and centrifugation to obtain labeled FMDV 146S antigen;

[0040] 3) the peripheral blood mononuclear cells obtained in step 1) are mixed with the labeled FMDV 146S antigen obtained in step 2), mouse anti-pig IgM-PE and mouse anti-pig IgG-FITC fluorescent antibody to obtain stained cells;

[0041] 4) Sorting FMDV-specific single B cells using multicolor flow cytometry;

[0042] 5) Reverse t...

Embodiment 1

[0062] 1. FMDV antigen labeling scheme

[0063] 1.1 O-type FMDV 146S antigen labeling scheme

[0064] The Type O FMDV 146S antigen was selected to be labeled using the Lightning-Link™ Rapid FluoProbes 647H Labeling Kit (InnovaBiosciences, San Diego, USA). First, the O-type FMDV 146S antigen was replaced in the PBS buffer with a 100kDa cut-off ultrafiltration tube. Centrifuge at 4000rpm for 10min to concentrate the antigen to 1mg / ml (OD 260 =7.6 is approximately equal to 1 mg / ml). Take 100μL 146S antigen and add 10μL LL-modifier reagent, mix gently, then pipette 100μL into a tube of Lightning-LinkTM mix reaction dye, gently suction to resuspend the powder, and react at room temperature (20-25℃) for 3 hours. Add 10 μL of LL-quencher reagent for 30 minutes to terminate the reaction. Then use a 100kDa ultrafiltration tube to centrifuge at 4000rpm for 10min to replace the labeled antigen into PBS buffer. Add an equal volume of 100% glycerol and store at -70°C. The labeled ant...

Embodiment 2

[0149] 1. FMDV Antigen Labeling Protocol

[0150] 1.1 Type A FMDV 146S antigen labeling scheme

[0151] Choose Pacific Blue TM Antibody labeling kit (Thermo Scientific, USA) labeled type A FMDV146S antigen. At first with cut-off 100kDa ultrafiltration tube A type FMDV 146S antigen is replaced in the PBS damping fluid, 4000rpm centrifugal 10min, concentrated antigen concentration is to 1mg / ml (OD 260 =7.6 is approximately equal to 1 mg / ml). Take 10 μL of sodium bicarbonate buffer (1M, pH=8.3) and add 100 μL of 146S antigen, mix gently, then draw 100 μL from it and add it to a tube of reaction dye, and turn it upside down 10 times to fully dissolve the dye. Incubate at room temperature (20-25°C) for 1 hour, and invert up and down 3 times every 10-15 minutes during this period. Then use a 100kDa ultrafiltration tube to centrifuge at 4000rpm for 10min to replace the labeled antigen into PBS buffer. Add an equal volume of 100% glycerol and store at -70°C. The labeled antigen ...

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Abstract

The invention relates to a preparation method of a pig-source single-chain genetic engineering antibody against foot-and-mouth disease viruses, and belongs to the technical field of preparation of antibodies. PBMCs are isolated from an immune pig body with a foot-and-mouth disease virus antigen, an FMDV 146 antigen is marked with a dyestuff, the PBMCs are dyed, and a single B cell combined with FMDV is sorted by virtue of a multicolor flow cytometer; combined with single-cell antibody gene sequencing, VH and VL genes are obtained respectively, and through a flexible linker and CHO-S expressionengineering antibody technology, the FMDV specific pig-source single-chain genetic engineering antibody is obtained through expression. The antibody obtained by the preparation method has the advantages of good genetic diversity, high efficiency, whole-host (pig) source, few needed cell quantity and the like.

Description

technical field [0001] The invention relates to the technical field of antibody preparation, in particular to a method for preparing an anti-foot-and-mouth disease virus pig-derived single-chain genetic engineering antibody. Background technique [0002] Foot-and-mouth disease (FMD) is a major animal disease that seriously harms pigs, cattle and sheep. There are seven serotypes of foot-and-mouth disease virus (FMDV), which are O, A, Asia 1, C, and SAT1-3, and there is no cross-immune reaction between each serotype. O-type FMDV and A-type FMDV are mainly prevalent in my country. Each serotype of FMDV is further divided into several topological types (VP1, 85% amino acid homology) according to the circulating regions. There are currently three lineages of O-type FMDV in China, namely O / Mya / 98 (Southeast Asian topological type), O / PanAsia (Central Southeast Asian topological type) and O / Cathay (Chinese topological type). The antigenic structures of the three lineage viruses ...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/85C12N5/078C12N15/13
CPCC07K16/1009C07K2317/35C07K2317/622C12N5/0634C12N15/85C12N2800/22
Inventor 李坤曹轶梅卢曾军刘在新孙普马雪青周莎莎朱国强白兴文李平花付元芳
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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