Site-directed Saturation Mutagenesis Library of Bacillus thuringiensis Cry1 Toxin-specific Single-chain Antibody Fragment by Homology Modeling and Molecular Docking
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Abstract:
There is a growing concern regarding potential risks to the ecology and food safety due to various Cry toxins from Bacillus thuringiensis (Bt) present in transgenic food. Identification of highly efficient broad-spectrum antibodies against multiple Cry toxins and development of rapid-detection methods are the basis for monitoring Bt transgenic crops to ensure food safety and protection of the ecological environment. Additionally, genetically engineered antibodies can easily undergo site-directed modification and can assume a structure similar to that of Cry toxins. Therefore, the three-dimensional structures of scFv and Cry1 toxins were analyzed via homology modelling, using human single-chain antibody fragment (scFv) against Bt Cry1Ac toxin previously developed by our group. Additionally, the key amino acid binding sites between scFv and Cry1 toxins were analyzed using molecular docking technique. Subsequently, site-directed modifications of the key amino acid binding sites and their adjacent sites using overlap-extension polymerase chain reaction (PCR) was carried out to construct a site-directed saturation mutagenesis library, to provide material for screening broad-spectrum antibodies to detect multiple Cry1 toxins.
