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Microcystin-LR nanobody screening from an alpaca phage display nanobody library and its expression and application

文献类型： 外文期刊

作者： Xu, Chongxin 1 ; Yang, Ying 1 ; Liu, Liwen 1 ; Li, Jianhong 1 ; Liu, Xiaoqin; Zhang, Xiao 2 ; Liu, Yuan 2 ; Zhang, Cun 1 ;

作者机构： 1.Nanjing Normal Univ, Coll Life Sci, Nanjing 210023, Jiangsu, Peoples R China

2.Jiangsu Acad Agr Sci, Key Lab Food Qual & Safety Jiangsu Prov, State Key Lab Breeding Base, Inst Food Safety & Nutr, Nanjing 210014, Jiangsu, Peoples R China

3.Jiangsu Acad Agr Sci, Key Lab Food Qual & Safety Jiangsu Prov, State Key Lab

关键词： Microcystin-LR; Phage display antibody library; Nanobody; Protein expression; ELISA

期刊名称：ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY （ 影响因子：6.291； 五年影响因子：6.393 ）

ISSN： 0147-6513

年卷期： 2018 年 151 卷

页码：

收录情况： SCI

摘要： Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC50) were 0.87, 1.17 and 1.47 mu g/L, their detection limits (IC10) were 0.06, 0.08 and 0.12 mu g/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7-116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0-106.5%, coefficient of variations (CVs) were 3.4-10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple MCs.

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