Functional Characterization of Anti-Parathion-ethyl single-chain variable fragment expressed in insect cells

• 1、Institute of Pesticide and Environmental Toxicology, Zhejiang University

Abstract：A functional single-chain variable fragment (scFv) gene specific to parathion-ethyl was expressed in Bac-to-Bac baculovirus expression system. The specific heavy and light variable chains were amplified from a hybridoma cell line 4C6 secreting monoclonal antibody (mAb) against parathion-ethyl and assembled as scFv fragments by splice overlap extension polymerase chain reaction (SOE-PCR). The scFv-4C6 gene constructed into expression plasmid (pFast-scFv) was transformed into E.coli DH10Bac to perform the transposition reaction. Then, the bacmid-scFv was isolated and transfected into Sf9 insect cells to generate the recombinant baculovirus. A small-scale expression of scFv-4C6 was carried out by infecting insect cells with baculoviral stock and confirmed by Western blotting. Finally, the recombinant scFv-8C10, with the relative molecular mass of approximately 28.3 kD, was successfully constructed and expressed in Sf9 cells, besides, the expression peak were appeared after 72 h transfection. As tested by ic-ELISA, the performance of scFv-4C6 were similar to those of the parental mAb, with a high sensitivity (IC50 value) of 7.9 ng/mL to parathion-ethyl and 12% cross reactivity to parathion-methyl. All above results demonstrated that the functional scFv-4C6 specific to parathion-ethyl was successfully expressed in insect cells.

Keywords： pesticide parathion single-chain variable fragment (scFv) antibody ELISA