农药学学报2016，Vol.18Issue(2)：177-184,8.DOI:10.16801/j.issn.1008-7303.2016.0023
抗对硫磷单链抗体在昆虫细胞中的表达及活性鉴定
Expression and characterization of single-chain variable fragment (scFv) antibody against parathion-ethyl in insect cells
摘要
Abstract
In this study, scFv-coding genes were obtained from a hybridoma cell and expressed in Bac-to-Bac baculovirus expression system, and the characterizations of scFv were verified by Western blotting, ELISA and compared with monoclonal antibody. Firstly, the specific heavy and light variable chains were amplified from hybridoma cell line 4C6, which secreting monoclonal antibody (mAb) against parathion-ethyl, and then they were assembled as scFv fragments by splice overlap extension polymerase chain reaction (SOE-PCR). Thereafter, the bacmid-scFv-4C6 was constructed and transfected into Sf9 insect cells to generate the recombinant antibody which was further verified by Western blotting. The results demonstrated that the recombinant scFv-4C6, with the relative molecular mass of approximately 28.3 kD, was successfully constructed and expressed in Sf9 cells. Besides, the expression peak was reached at 72 h after the transfection. As tested by ic-ELISA, the performances 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, 12% cross reactivity to parathion-methyl, and 1.8% cross reactivity to fenitrothion.关键词
对硫磷/单链抗体/昆虫细胞表达/酶联免疫吸附Key words
parathion-ethyl/single-chain variable fragment (scFv) antibody/insect cell expression system/ELISA分类
农业科技引用本文复制引用
刘蕊,项丹丹,刘鹏琰,梁晓,郭逸蓉,朱国念..抗对硫磷单链抗体在昆虫细胞中的表达及活性鉴定[J].农药学学报,2016,18(2):177-184,8.基金项目
公益性行业(农业)科研专项(201203094-3) （农业）
