title: Construction of a Single Chain Variable Fragment Antibody (scFv) against Carbaryl and Its Interaction with Carbaryl.
abstract: Carbaryl is a low molecular weight insecticide that inhibits cholinesterase. Residues of carbaryl in food and the environment have damaged human health. A high-speciﬁcity scFv that can identify carbaryl is still lacking. In the present study, an anti-carbaryl scFv gene was prepared by cloning VL and VH genes from hybridoma cells secreting monoclonal antibody, then VH and VL were fused together using splicing by overlap extension (SOE) PCR with a flexible polypeptide linker connector (Gly4Ser)3, and then the scFv-pET-26b recombinant plasmid was constructed and transformed into E. coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was identified by SDS-PAGE and ELISA. The three-dimensional structure of the anti-carbaryl scFv was constructed by computer modeling, and carbaryl was docked to the scFv model to obtain the structure of the binding complex. The binding site was composed of Ala51, Ser52, Ile51, Gly54, Ser56, Arg98, and Gly100. This helps to understand the mechanism of interaction between anti-carbaryl antibody and antigen. Furthermore, it provides guidance for in vitro affinity maturation of anti-carbaryl antibody.
authors.affiliation: Hebei North University, Zhangjiakou, Hebei, 075000, China. zhangxiuyuan917@163.com.
authors.forename: Zhang
authors.initials: Z
authors.lastname: Xiuyuan
authors.forename: Huang
authors.initials: H
authors.lastname: Zhihong
authors.forename: Wang
authors.initials: W
authors.lastname: Lixia
authors.forename: Liu
authors.initials: L
authors.lastname: Xiaonan
mesh_terms: D012721:Carbaryl / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006801:Humans; D007306:Insecticides / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D007306:Insecticides; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D012721:Carbaryl