Production and Characterization of a Single-Chain Variable Fragment Linked Alkaline Phosphatase Fusion Protein for Detection of O,O-Diethyl Organophosphorus Pesticides in a One-Step Enzy

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• 作者：Zhen-Lin Xu ; Jie-Xian Dong ; Hong Wang ; Zhen-Feng Li ; Ross C. Beier ; Yue-Ming Jiang ; Hong-Tao Lei ; Yu-Dong Shen ; Jin-Yi Yang ; Yuan-Ming Sun
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2012
• 出版时间：May 23, 2012
• 年：2012
• 卷：60
• 期：20
• 页码：5076-5083
• 全文大小：337K
• 年卷期：v.60,no.20(May 23, 2012)
• ISSN：1520-5118

文摘

A single-chain variable fragment (scFv) linked alkaline phosphatase (AP) fusion protein for detection of O,O-diethyl organophosphorus pesticides (O,O-diethyl OPs) was produced and characterized. The scFv gene was prepared by cloning V_L and V_H genes from hybridoma cells secreting monoclonal antibody with broad specificity for O,O-diethyl OPs. The amplified V_L and V_H regions were assembled using a linker (Gly₄Ser)₃ by means of splicing overlap extension polymerase chain reaction to obtain the scFv gene, which was cloned into the expression vector pLIP6/GN containing an AP gene to produce the scFv鈥揂P fusion protein in Escherichia coli strain BL21. The protein was purified by antigen-conjugated immunoaffinity chromatography and characterized by sodium dodecyl sulfate鈥損olyacrylamide gel electrophoresis, Western blotting, and competitive direct enzyme-linked immunosorbent assay (cdELISA). The fusion protein is bifunctional, retaining both antigen binding specificity and AP enzymatic activity. Analysis of spiked and blind river water and Chinese cabbage samples demonstrated that the fusion protein based cdELISA_FP exhibited good sensitivity and reproducibility.

Keywords:

single-chain variable fragment; alkaline phosphatase; fusion protein; broad specificity; organophosphorus pesticide