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 Enzyme-Linked Immunosorbent Assay
2012
Xu, Zhen-Lin | Dong, Jie-Xian | Wang, Hong | Li, Zhen-Feng | Beier, Ross C. | Jiang, Yue-Ming | Lei, Hong-Tao | Shen, Yu-Dong | Yang, Jin-Yi | Sun, Yuan-Ming
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 VL and VH genes from hybridoma cells secreting monoclonal antibody with broad specificity for O,O-diethyl OPs. The amplified VL and VH 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–AP fusion protein in Escherichia coli strain BL21. The protein was purified by antigen-conjugated immunoaffinity chromatography and characterized by sodium dodecyl sulfate–polyacrylamide 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 cdELISAFP exhibited good sensitivity and reproducibility.
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