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Anti-African swine fever virus single-chain antibody as well as preparation method and application thereof

An African swine fever virus, single-chain antibody technology, applied in the field of single-chain antibodies

Active Publication Date: 2020-08-21
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Looking back at the development of the antibody field in recent years, we can see that traditional antibodies can no longer meet the needs of the current antibody market. People are in urgent need of a flexible antibody that is easy to prepare, strong in specificity, high in stability, and small in molecular weight. Efficiently act on specific parts

Method used

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  • Anti-African swine fever virus single-chain antibody as well as preparation method and application thereof
  • Anti-African swine fever virus single-chain antibody as well as preparation method and application thereof
  • Anti-African swine fever virus single-chain antibody as well as preparation method and application thereof

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Embodiment 1

[0097] Embodiment 1 Anti-African swine fever virus ScFv antibody (VH-VL κ6 ) preparation and its gene sequence

[0098] 1. Materials and methods

[0099] 1.1 Materials

[0100] 1.1.1 Test animals

[0101] 1 African swine fever immune resistant pig (5 months old, Xinxiang Pig Farm, Henan).

[0102] 1.1.2 Strains and reagents

[0103] LTS1110 Porcine Peripheral Blood Lymphocyte Separation Kit was purchased from Tianjin Haoyang Biological Products Company; PMD TM 20-T carrier, E.coli JM109 competent, HS DNA Polymerase, RNase-free Water, DL2,000 DNA Marker, and 6×Loading Buffer were all purchased from Bao Biological Company; BL21(DE3) competent was purchased from Beijing Quanshijin; Trizol, nickel affinity chromatography resin, and DNA fragment recovery Kits and plasmid extraction kits are OMEGA products; molecular biology reagents are from Sigma; other biochemical reagents are of domestic analytical grade.

[0104] 1.2 Method

[0105] 1.2.1 Lymphocyte separation

[010...

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Abstract

The invention discloses an anti-African swine fever virus single-chain antibody as well as a preparation method and application thereof. According to the invention, lymphocytes are separated from peripheral blood of naturally infected African swine fever immune tolerant pigs; extracting is carried out to obtain the total mRNA of the separated lymphocytes; the total cDNA fragments are obtained through an RT-PCR method; cDNA is taken as a template, under the action of a corresponding primer with a Linker joint, swine ScFv antibody gene sequence is obtained through an SOE-PCR method; constructingof an ScFv antibody gene sequence to a pET-30a vector is carried out, and BL21 competent cell transformation is carried out; the ScFv antibody (VH-VL kappa 6) aiming at the African swine fever virusis screened from a single colony after transformation, and preliminary activity identification is carried out on the screened ScFv antibody (VH-VL kappa 6) by adopting an ELISA test, it shows that theantibody has African swine fever reaction activity. The invention provides a new material for early diagnosis, prevention and control of African swine fever, and provides a new technical means for controlling epidemic situation propagation of African swine fever as soon as possible.

Description

technical field [0001] The invention relates to a single-chain antibody against African swine fever virus. In addition, the present invention also relates to the preparation method and application of the antibody. The invention belongs to the field of biotechnology. Background technique [0002] African swine fever (African swine fever, ASF) is a highly fatal hemorrhagic disease of pigs caused by African swine fever virus (African swine fever Virus, ASFV), which is highly contagious. ASFV is an icosahedral double-stranded DNA virus with a diameter of 200 nm. It is the only member of the African swine fever virus family and the only known vector-borne virus. African swine fever first broke out in Kenya, Africa in 1921, and was introduced to my country in August 2018. As of February 2019, African swine fever has occurred in 24 provinces across the country, causing huge economic losses to the pig industry. At present, there is no effective vaccine against this virus in the w...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13C12N15/70G01N33/569G01N33/68C12R1/19
CPCC07K16/081C07K2317/14C07K2317/20C07K2317/622C12N15/70G01N33/56983G01N33/6854G01N2333/01G01N2469/10
Inventor 郑海学王丽娟田宏石正旺罗俊聪杨帆
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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