Construction of scFv Antibody for the Determination of Cry1A Toxins and Preliminary Study on Their Interaction Difference
QU Ting-Ting1,2, ZHANG Xiao2, DONG Sa2, LIU Bei-Bei2, LI Pan2, WANG Yun2, ZHANG Cun-Zheng2, LIU Xian-Jin1,2,*
1 College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; 2 Key Laboratory of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base/Key Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of Agriculture / Institute of Food Quality Safety and Detection Research, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
Abstract：To establish a cost-effective detection method for Cry1 toxins, a hybridoma cell line 2D10 was used as a cDNA template to construct a single-chain antibody (scFv) gene via gene splicing by overlap extension PCR (SOE-PCR). After being transferred into Escherichia coli BL21 (DE3), the scFv gene was expressed and a double antibody sandwich ELISA (DAS-ELISA) was established. Meanwhile, the key factors affecting the interaction of scFv to Cry1A toxins were analyzed by molecular docking simulation. As a result, the scFv gene was successfully constructed and the scFv antibody with high activity was purified (about 28 kD). The minimum detection limit (LOD) of the established DAS-ELISA for Cry1Ab/Cry1Ac toxin was 20 ng/mL. The docking results showed that the three-dimensional structure of the toxin determined its binding activity, hydrogen bonds and hydrophobic interaction played an important role during the binding process between scFv and Cry1A toxins. In this study, a scFv was constructed and expressed based on genetic engineering antibody technology, a DAS-ELISA was established for Cry1Ab/Cry1Ac toxin determination and the recognization mechanism difference of scFv to Cry1Ac/Cry1Ab and Cry1Aa was preliminarily analyzed which provide a theoretical basis for the research and development of a new broad-spectrum scFv.
曲婷婷, 张霄, 董飒, 刘贝贝, 李盼, 王耘, 张存政, 刘贤金. 单链抗体对Cry1A类毒素的检测方法建立及识别差异初步研究[J]. 农业生物技术学报, 2019, 27(3): 516-525.
QU Ting-Ting, ZHANG Xiao, DONG Sa, LIU Bei-Bei, LI Pan, WANG Yun, ZHANG Cun-Zheng, LIU Xian-Jin. Construction of scFv Antibody for the Determination of Cry1A Toxins and Preliminary Study on Their Interaction Difference. 农业生物技术学报, 2019, 27(3): 516-525.
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