Production and characterization of a single-chain variable fragment antibody from a site-saturation mutagenesis library derived from the anti-Cry1A monoclonal antibody
文献类型： 外文期刊
作者： Dong, Sa 1 ; Gao, Meijing 1 ; Bo, Zongyi 3 ; Guan, Lingjun 2 ; Hu, Xiaodan 1 ; Zhang, Hanxiaoya 1 ; Liu, Beibei 1 ; Li, P 1 ;
作者机构： 1.Jiangsu Acad Agr Sci, Inst Food Safely & Nutr, Key Tab Food Qual & Safety Jiangsu Prov,Minist Ag, State Key Lab Breeding Base,Key Lab Control Techn, Nanjing 210014, Peoples R China
2.Yangzhou Univ, Coll Hort & Plant Protect, Yangzhou 225009, Jiangsu, Peoples R China
3.Nanjing Agr Univ, Coll Vet Med, Nanjing 210095, Jiangsu, Peoples R China
关键词： Cry1A toxins; ScFy antibody; Site-saturation mutagenesis
期刊名称：INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES （ 影响因子：6.953； 五年影响因子：6.737 ）
ISSN： 0141-8130
年卷期： 2020 年 149 卷
页码：
收录情况： SCI
摘要： There are plenty of applications of Cry1A toxins (Cry1Aa, Cry1Ab, Cry1Ac) in genetically modified crops, and it is necessary to establish corresponding detection methods. In this study, a single-chain variable fragment (scFv) with high affinities to Cry1A toxins was produced. First, the variable regions of heavy (V-H) and light chain (V-L) were amplified from hybridoma cell 5B5 which secrete anti-Cry1A monoclonal antibody (mAb) and then spliced into scFv-5B5 by overlap extension polymerase chain reaction (SOE-PCR). Subsequently, site-saturation mutagenesis was performed after homology modeling and molecular docking, which showed that asparagine(35), phenylalanine(36), isoleucine(104), tyrosine(105), and serine(196), respectively, located in V-H complementarity-determining region (CDR1 and CDR3) and V-L framework region (FR3) were key amino acid sites. Then, the mutagenesis scFv library (1.35 x 10(5) CFU/mL) was constructed and a mutant scFv-2G12 with equilibrium dissociation constant (KD) value of 9.819 x 10(-9) M against Cry1Ab toxin, which was lower than scFv-5B5 (2.025 x 10(-8) M) was obtained by biopanning. Then, enzyme-linked immunosorbent assay (ELISA) was established with limit of detection (LOD) and limit of quantitation (LOQ) of 4.6-9.2 and 11.1-17.1 ng mL(-1) respectively for scFv-2G12, which were lower than scFv-5B5 (12.4-22.0 and 23.6-39.7 ng mL(-1)). Results indicated the promising prospect of scFv-2G12 used for the detection of Cry1A toxins. (C) 2020 Elsevier B.V. All rights reserved.
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