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Disulfide-stabilized anti-chloramphenicol single-chain antibody as well as construction method and application thereof

A single-chain antibody and chloramphenicol technology, applied in chemical instruments and methods, specific peptides, and the use of vectors to introduce foreign genetic material, etc., can solve problems such as poor stability, improve stability, and solve low-level expression effects

Inactive Publication Date: 2011-01-05
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The object of the present invention is to provide a disulfide bond-stabilized antibody based on the lack of disulfide bonds and poor stability between the variable regions existing in the existing single-chain antibody for detecting chloramphenicol residues. Chloramphenicol scFv

Method used

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  • Disulfide-stabilized anti-chloramphenicol single-chain antibody as well as construction method and application thereof
  • Disulfide-stabilized anti-chloramphenicol single-chain antibody as well as construction method and application thereof
  • Disulfide-stabilized anti-chloramphenicol single-chain antibody as well as construction method and application thereof

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Embodiment 2

[0024] The construction of the anti-chloramphenicol single chain antibody of embodiment disulfide bond stabilization

[0025] 1. Materials and methods

[0026] 1.1 Materials

[0027] Anti-CAP Mab crude product, containing anti-CAP V H (FJ477893) and V L (FJ477894) gene plasmid pMD18-T-V H with pMD18-T-V L , chloramphenicol-ovalbumin conjugate (CAP-OVA), Escherichia coli DH5α, etc. were all prepared or preserved by our laboratory. Escherichia coli BL21(DE3), expression vectors pET21a and pET32a are all products of NOVAGEN, pMD18-T, Taq enzyme, restriction endonuclease, T4 DNA ligase, DNA ladder, TaKaRa MutanBEST Kit, etc. were all purchased from Dalian Bao Biological Engineering Co., Ltd. Co., Ltd., the DNA recovery kit was purchased from Biotek Biotechnology Co., Ltd., HisTrap FF column (1ml), HiTrap Protein G column (1ml), HRP-anti-His6 tag and HRP-goat-anti-mouse IgG were all GE companies The product, BCA kit is the product of Guangzhou Meijin Biotechnology Co., Ltd. ...

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Abstract

The invention discloses a disulfide-stabilized anti-chloramphenicol single-chain antibody as well as a construction method and application thereof. The amino-acid sequence of the anti-chloramphenicol single-chain antibody is shown as SEQ ID NO:1. The construction method comprises the following steps of: by using the variable region gene of the anti-chloramphenicol antibody as a template, respectively adding a cysteine site-directed mutation in the variable regions of a light chain and a heavy chain through primer mutation PCR (Polymerase Chain Reaction), assembling into a disulfide-stabilized anti-chloramphenicol single-chain antibody gene through overlap extension PCR, introducing synonymous mutation into the 5'-terminal 30bp sequence of the gene through the primer mutation PCR, and then transducing the gene into a colon bacillus expression system to obtain expressed protein, i.e. the disulfide-stabilized anti-chloramphenicol single-chain antibody. The construction form of a pair of disulfide bonds is introduced on the basis of the single-chain antibody into the anti-chloramphenicol single-chain antibody by simulating the structure of a natural antibody, and the stability of the antibody can be improved. The disulfide-stabilized anti-chloramphenicol single-chain antibody can replace a monoclonal antibody to be used for chloramphenicol residue detection.

Description

technical field [0001] The invention relates to the field of chloramphenicol antibodies, in particular to a disulfide bond-stabilized anti-chloramphenicol single-chain antibody and its construction method and application. Background technique [0002] Chloramphenicol (CAP) is an antibiotic that can cause serious diseases such as human aplastic anemia and agranulocytosis. It has been banned by the United Nations since 1994. my country has also clearly stipulated that chlorine in animal-derived foods has been regulated since 2002. Mycin residues cannot be detected (Wang Yiqin, Bao Yongyong, Hu Lieshan, etc. Application of ELISA in detecting chloramphenicol residues in animal tissues [J]. China Agricultural Bulletin, 2006, 22(4): 26-29.), but Due to its low cost and high efficiency, it is still widely illegally abused in the breeding industry, which brings great challenges to people's life and health and the export trade of my country's animal-derived food-related products. Spec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N15/11C12N15/70G01N33/53
Inventor 李黄金陈伟赵林柴伟佳唐伟
Owner GUANGDONG PHARMA UNIV
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