Construction, Expression, and Identification of Double Light Chain (V-L-V-L) Antibody from a Unique Bt Cry1-Specific Monoclonal Antibody

文献类型： 外文期刊

第一作者： Dong, Sa

作者： Dong, Sa;Gao, Meijing;Zhang, Hanxiaoya;Wang, Yulong;Liu, Beibei;Li, Pan;Liu, Xianjin;Zhang, Cunzheng;Dong, Sa;Guan, Lingjun;Qiao, Kang

作者机构：

关键词： Cry1 toxins; ScFv antibody; Molecular docking; Binding activity; V-L-V-L antibody

期刊名称：FOOD ANALYTICAL METHODS （ 影响因子：3.366； 五年影响因子：3.07 ）

ISSN： 1936-9751

年卷期： 2020 年 13 卷 8 期

页码：

收录情况： SCI

摘要： Using the hybridoma cells 2D3 which secrete anti-Cry1 monoclonal antibody as DNA source, the variable region genes of heavy chain (V-H) and light chain (V-L) were amplified and assembled as single-chain variable fragment (scFv). The scFv-2D3 genes were cloned into pET-26b vectors for expression and scFv-2D3 expression was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot (WB). The activity of scFv-2D3 was determined by indirect enzyme-linked immune sorbent assay (ELISA). As a result, scFv-2D3 was successfully constructed and expressed, with specific recognition activities to the Cry1 toxins. Homologous modeling and molecular docking results showed that V-L was the main combination domain of scFv-2D3 with Cry1 toxins. Therefore, V-H and V-L were separately expressed and then assayed by indirect ELISA, indicating that both V-H and V-L could recognize the Cry1 toxin, and the activity of V-L was higher than that of V-H, which confirming the molecular docking result. Afterward, double light chain antibodies (V-L-V-L) were designed and produced, and the binding activities of V-L-V-L against Cry1Aa, Cry1Ab, and Cry1Ac were significantly enhanced with OD450 values improved 1.18, 1.44, and 1.22 times, respectively, compared with scFv-2D3. For V-L-V-L-based double antibody sandwiched ELISA (DAS-ELISA), the limits of detection (LOD) and limits of quantification (LOQ) were 10.5-16.0 and 12.8-21.5 ng mL(-1) for seven Cry1 toxins, respectively, which were lower than scFv-2D3 (14.8-29.2 and 39.0-50.3 ng mL(-1)). The results were beneficial to developing high-throughput and high-sensitive immune-detecting technology for Cry1 toxins.

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