Broad specificity immunoassay for detection of Bacillus thuringiensis Cry toxins through engineering of a single chain variable fragment with mutagenesis and screening.
Broad specificity immunoassay for detection of Bacillus thuringiensis Cry toxins through engineering of a single chain variable fragment with mutagenesis and screening.
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DOI：
10.1016/j.ijbiomac.2017.09.058
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发表时间：
2018-02
影响因子：
8.2
通讯作者：
J. Zhong;Xiaodan Hu;Xiao Zhang;Yuan Liu;Chongxin Xu;Cunzheng Zhang;Manman Lin;Xianjin Liu
中科院分区：
文献类型：
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作者：
J. Zhong;Xiaodan Hu;Xiao Zhang;Yuan Liu;Chongxin Xu;Cunzheng Zhang;Manman Lin;Xianjin Liu
在世界范围内，转基因作物的种植已经大大增加。目前已有超过10种苏云金芽孢杆菌毒素被应用于转基因作物中，因此有必要开发一种高效且经济实惠的检测方法，同时筛选作物和食品中具有相似结构的此类化合物。在这里，我们描述了一种使用位点定向诱变的方法，该方法增强了单链可变片段（scFv）的一般特异性。在对构建的诱变文库中的混合抗原（Cry1Ab、Cry1Ac、Cry1B、Cry1C和Cry1F）进行三轮筛选后，一个突变体显示出与一系列Cry毒素结合的更强能力。将噬菌体突变体D9克隆到pET26b表达载体上，进行诱导纯化。利用突变体D9建立了一种间接竞争免疫分析法，成功识别了5种靶向Cry1毒素，其工作范围为0.20 ~ 2.22 μg mL−1。5种Cry毒素加样回收率在86.67% ~ 96.67%之间，变异系数小于8%。结果表明，基于scFv的免疫分析是一种具有广泛特异性的有前途的检测Cry毒素的方法。
The cultivation of genetically modified crops (GMCs) has greatly increased worldwide. Given the fact that over 10Bacillus thuringiensisCry toxins have been applied in GMCs, there is a need to develop an efficient and economically affordable detection method that simultaneously screen such compounds with similar structures in crops and foodstuff. Here we described an approach using a site-directed mutagenesis that enhances the generic specificity of single chain variable fragment (scFv). After three rounds of panning against mixed antigen (Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F) from the constructed mutagenesis library, one mutant showed greater ability to bind to a range of Cry toxins. The phage mutant, named D9, was cloned into pET26b expression vector, and then induced and purified. The mutant D9 was used to develop an indirect competitive immunoassay that sucessfully recognizes five targeted Cry1 toxins with a working range from 0.20 to 2.22 μg mL−1. The recoveries of five Cry toxins from spiked rice samples ranged from 86.67% to 96.67%, with a coefficient of variation less than 8%. The results suggest that the immunoassay based on a scFv is a promising approach for detection of Cry toxins with a broad specificity.