title: Expression of V(H)-linker-V(L) orientation-dependent single-chain Fv antibody fragment derived from hybridoma 2E6 against aflatoxin B1 in Escherichia coli.
abstract: Aflatoxin B1 (AFB1) is a toxic secondary metabolic product, which threatens human and animal health. Antibody is a key factor for immunoassay against toxic stuff like AFB1, and single-chain Fv antibody fragment (scFv) has become a popular format of genetically engineered antibody. In this study, four hybridoma cell lines against AFB1 were obtained, and then scFvs 2E6 derived from hybridoma cell line 2E6 were constructed in different V(H)/V(L) orientations. Subsequently, scFvs 2E6 were expressed in E. coli BL21(DE3) mainly in the form of inclusion body. SDS-PAGE, Western blot and ELISA were employed to characterize scFvs 2E6. The results revealed that the yield of inclusion body of scFvs 2E6 in either V(H)/V(L) orientation was similar; however, only the scFv in V(H)-linker-V(L) orientation showed anti-AFB1 bioactivity after refolding. The present study underscores the importance of choosing optimal V(H)/V(L) orientation for scFv construction, and scFv may be favorable for immunoassays in food industry.
authors.affiliation: College of Food Science and Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei, People's Republic of China, aip.liu@hotmail.com.
authors.forename: Aiping
authors.initials: A
authors.lastname: Liu
authors.forename: Yang
authors.initials: Y
authors.lastname: Ye
authors.forename: Weifeng
authors.initials: W
authors.lastname: Chen
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Wang
authors.forename: Fusheng
authors.initials: F
authors.lastname: Chen
mesh_terms: D016604:Aflatoxin B1 / Q000737:chemistry*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D006825:Hybridomas; D002479:Inclusion Bodies / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D057127:Single-Chain Antibodies / Q000096:biosynthesis*
chemical_list: D057127:Single-Chain Antibodies; D016604:Aflatoxin B1