基于同源建模与分子对接技术构建抗Bt Cry1类毒素单链抗体定点饱和突变库

Site-directed Saturation Mutagenesis Library of Bacillus thuringiensis Cry1 Toxin-specific Single-chain Antibody Fragment by Homology Modeling and Molecular Docking

摘要:

转基因食品中苏云金芽胞杆菌（Bt）Cry毒素种类较多，对生态环境和食品安全等造成的潜在风险日益受到关注，制备针对多种Cry毒素的高效广谱抗体，进而建立广谱性快速检测方法，是对转Bt基因作物进行监管并确保生态环境和食品安全的基础。本研究利用基因工程抗体易于定向修饰及Cry毒素具有相似结构的特点，以实验室前期获得的人源化抗苏云金芽胞杆菌（Bt）Cry1Ac毒素单链抗体为材料，通过同源建模模拟单链抗体及五种Cry1类毒素的三维结构并利用分子对接技术分析单链抗体与Cry1类毒素结合的关键氨基酸位点；在此基础上利用重叠延伸PCR技术对关键氨基酸位点及其邻近位点进行定向改造，以构建定点饱和突变库，为筛选检测多种Cry1类毒素的广谱特异性抗体提供实验材料。

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.