Construction, expression and functional analysis of anti-clenbuterol codon-optimized scFv recombinant antibody.

Qi Lu, Yao-Yao Hou, Xi-Xia Liu, Hong Wang, Jian-Jun Hou, Jing-Li Wei, Shan-Shan Zhou, Xin-Ya Liu

Journal: Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 2020;135():110973

PMID: 31738983

Full Text on Elsevier Science View on PubMed

Abstract

The construction, expression and functional analysis of codon-optimized single-chain variable fragment (coscFv) against clenbuterol (CBL) prepared from the Escherichia coli system is described. First, the ionic concentration for coscFv expression was optimized through single-factor experiments. Then, the extraction conditions of inclusion bodies were optimized, and coscFv was affinity-purified. Finally, the functional analysis of coscFv was elucidated by indirect competitive enzyme-linked immunosorbent assay (icELISA) and molecular docking. After optimizing the ionic concentration, the yield of coscFv increased from 21.69% to 23.26%. The molecular weight of coscFv was determined to be approximately 27 kDa according to the SDS-PAGE and Western blot assay. The percentage of coscFv was as high as 43.9% after the inclusion bodies were extracted, washed, and dissolved. Functional analysis indicated that the coscFv recognized CBL, and the 50% inhibition average concentration of CBL (IC) was 4.22 ± 0.01 (n = 3) ng/mL. The binding site between coscFv and CBL consisted of Asp33H, Met34H, Ser50H, Arg52H, Tyr57H, Leu59H, Asp99H, and Tyr93L. Our study confirms that coscFv can bind with CBL through the key amino acid residues and can be used to sensitively detect CBL.

Address: Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Huangshi, 435002, China; National Demonstration Center for Experimental Biology Education, Hubei Normal University, Huangshi, 435002, China.; Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Huangshi, 435002, China; National Demonstration Center for Experimental Biology Education, Hubei Normal University, Huangshi, 435002, China. Electronic address: [email protected].; Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou, 510642, China. Electronic address: [email protected].

DOI: 10.1016/j.fct.2019.110973

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MeSH Terms: Adrenergic beta-Agonists, Amino Acid Sequence, Binding Sites, Clenbuterol, Cloning, Molecular, Enzyme-Linked Immunosorbent Assay, Escherichia coli, Molecular Docking Simulation, Protein Binding, Recombinant Proteins, Sequence Alignment, Single-Chain Antibodies