Microcystin-LR heterologous genetically engineered antibody recombinant and its binding activity improvement and application in immunoassay

文献类型： 外文期刊

第一作者： Xu, Chongxin

作者： Xu, Chongxin;He, Dan;Hao, Jia;Xu, Chongxin;Zu, Yao;Hong, Sujuan;Li, Jianhong;Hao, Jia

作者机构：

关键词： Microcystin-LR; Genetically engineered antibody; Chain shuffling; Site-directed mutagenesis; Immunoassay

期刊名称：JOURNAL OF HAZARDOUS MATERIALS （ 影响因子：10.588； 五年影响因子：10.129 ）

ISSN： 0304-3894

年卷期： 2021 年 406 卷

页码：

收录情况： SCI

摘要： Microcystin-LR (MC-LR) is a high-toxic biohazard that pollutes ecological environment and agroproducts. In this study, a newly recombined genetically engineered antibody (AV(HH)-MVH) with higher thermal stability and binding activity was designed by chain shuffling and based on our previously obtained anti-MC-LR scFv and nanobody. Based on AV(HH)-MVH template, a capacity of 8.99 x 10(5) CFU/mL of phage display AV(HH)-MVH mutagenesis library was constructed by site-directed mutagenesis in MVH-CDR3 region, and then used for ultrasensitive mutants screening. Afterwards, a total of five positive AV(HH)-MVH mutants were isolated from the mutagenesis library, and their binding activity was higher than AV(HH)-MVH for MC-LR. The AV(HH)-MVH mutant 3 was cloned into pET-25b vector for soluble expression, and the concentration of target protein expressed in culture system was 43.5 mg/L. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was established based on purified AV(HH)-MVH mutant 3 protein, and it showed ultrasensitive binding activity for MC-LR with the detection limit of 0.0075 mu g/L, which was far below the maximum residue limit standard of 1.0 mu g/L in drinking water proposed by World Health Organization. The established IC-ELISA shows good accuracy, repeatability, stability and applicability for MC-LR spiked samples, and it is promising for MC-LR ultrasensitive monitoring.

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