Jiao Lingxia, Liu Yuan, Zhang Xiao, Liu Beibei, Zhang Cunzheng, Liu Xianjin
Key Laboratory of Food Quality and Safety of Jiangsu Province, Nanjing, 210014, China.
School of Food Science, Henan Institute of Science and Technology, Xinxiang, Henan, 453003, China.
Appl Microbiol Biotechnol. 2017 Aug;101(15):6071-6082. doi: 10.1007/s00253-017-8347-9. Epub 2017 Jun 11.
Potential ecological environmental and food safety risks of various Cry toxins of Bacillus thuringiensis (Bt) in transgenic food have received gradually increasing attention, which urged to establish an efficient and broad-spectrum detection technology for Cry toxins. Based on the single-domain antibody (sdAb) A8 against Bt Cry1Ab toxin screened from the humanized domain antibody library, the key amino acids of sdAb (A8) binding five kinds of Cry1 toxins were predicted using homology modeling and molecular docking technology, and the results showed that 105th asparagine, 106th arginine, 107th valine, and 114th arginine, respectively, located in heavy-chain complementarity-determining region 3 were common key amino acid sites. Subsequently, site-saturation cooperative mutagenesis of the four key sites was performed using overlap extension PCR, and multiple site-saturation mutagenesis sdAb library with the capacity of 1.2 × 10 colony-forming units (CFU) was successfully constructed. With alternating five Cry1 toxins as coating antigen, two generic sdAbs (2-C1, 2-C9) were screened out from the mutagenesis library, which could detect six kinds of Cry1 toxins at least. Through ELISA analysis, the binding activity of 2-C9 was significantly enhanced, and its OD values versus Cry1Aa, Cry1Ab, Cry1B, Cry1C, and Cry1E increased to 1.34, 1.53, 1.82, 2.39, and 2.7 times, respectively, compared with maternal antibody A8. The IC50 values of 2-C9 against Cry1Aa, Cry1Ab, Cry1B, and Cry1C were lower than that of A8, which showed that the affinity of 2-C9 against Cry1 toxins was enhanced. The results were beneficial to developing high-throughput and high-sensitive immune-detecting technology for Cry toxins.
转基因食品中苏云金芽孢杆菌（Bt）各种Cry毒素潜在的生态环境及食品安全风险已逐渐受到越来越多的关注，这促使人们建立一种高效、广谱的Cry毒素检测技术。基于从人源化结构域抗体库中筛选出的针对Bt Cry1Ab毒素的单结构域抗体（sdAb）A8，利用同源建模和分子对接技术预测了sdAb（A8）与5种Cry1毒素结合的关键氨基酸，结果表明分别位于重链互补决定区3的第105位天冬酰胺、第106位精氨酸、第107位缬氨酸和第114位精氨酸是共同的关键氨基酸位点。随后，利用重叠延伸PCR对这4个关键位点进行位点饱和协同诱变，成功构建了库容为1.2×10菌落形成单位（CFU）的多位点饱和诱变sdAb文库。以交替的5种Cry1毒素作为包被抗原，从诱变文库中筛选出2种通用sdAb（2-C1、2-C9），它们至少能检测6种Cry1毒素。通过ELISA分析，2-C9的结合活性显著增强，与母本抗体A相比，其对Cry1Aa、Cry1Ab、Cry1B、Cry1C和Cry1E的OD值分别提高到1.34、1.53、1.82、2.39和2.7倍。2-C9对Cry1Aa、Cry1Ab、Cry1B和Cry1C的IC50值低于A8，表明2-C9对Cry1毒素的亲和力增强。这些结果有利于开发用于Cry毒素的高通量、高灵敏度免疫检测技术。
