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KMID : 1094720160210010134
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Biotechnology and Bioprocess Engineering
2016 Volume.21 No. 1 p.134 ~ p.143
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Production of a phage-displayed mouse ScFv antibody against fumonisin B1 and molecular docking analysis of their interactions
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Hu Zu-Quan
Li He-Ping
Liu Jin-Long
Xue Sheng
Gong An-Dong
Zhang Jing-Bo
Liao Yu-Cai
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Abstract
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Fumonisins produced by Fusarium pathogens are mycotoxins present in maize and other grains in the field as well as during storage worldwide and pose a serious threat to humans and domestic animals. Fumonisin B consists of different chemotypes, and fumonisin B1 (FB1) is the most predominant fumonisin found in food/feed commodities. Recombinant antibody can be deployed to analyze the fumonisin toxicological mechanism and develop a simple and cost-effective method for the detection of fumonisins, which is vitally important for monitoring and preventing fumonisins from entering food/feed chains. In this study, FB1 conjugated to keyhole limpet hemocyanin was used to immunize mice, from which RNA was isolated to construct a recombinant antibody library. Successive panning of the library by phage display was used to select monoclonal phage clones reactive to FB1 conjugated to bovine serum albumin. Subsequent phage ELISA and sequencing analyses revealed four different reactive scFv antibodies specific to FB1. Soluble expression and ELISA analysis showed that one scFv antibody, FBMA1, had the highest reactivity and could be purified from bacterial cells in large quantities. Surface plasmon resonance measurements further revealed that the FBMA1 scFv antibody had a binding kinetics of K D = 1.89 × 10?7 M. Molecular modeling and docking analyses suggested that the FBMA1 antibody shaped a proper cavity to embed the whole FB1 molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic studies of intermolecular interactions and fumonisin toxicity, and for the development of an immunoassay for fumonisin-contaminated food/feed samples.
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KEYWORD
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fumonisin B1, single-chain variable fragment (scFv), phage display, binding kinetics, molecular docking
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