利用噬菌体展示技术筛选Cry2Ab毒素受体表位的方法
Using phage display to map the binding epitope of the Bacillus thuringiensis Cry2Ab toxin
齐佳，刘晨曦，吴孔明**
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作者单位：中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室北京100193
中文关键词：噬菌体展示，表位筛选，Cry2A
英文关键词：phage display，epitope mapping，Cry2Ab
中文摘要：苏云金芽孢杆菌(Bacillus thuringiensis，Bt)产生的内毒素具有杀虫活性，Cry2Ab毒素作为 Bt棉花的杀虫活性蛋白，其在靶标昆虫体内的结合受体及作用位点尚不清楚，本研究采用噬菌体展示(phage display)的方法，经 4轮的“吸附 —洗脱—扩繁”筛选，并对阳性克隆所携带的外源 DNA片段进行序列测定后，得到 2段能够与活化 Cry2Ab毒素相互作用的多肽序列，通过酶联免疫结合试验(ELISA)进一步证明，这 2段多肽序列与活化 Cry2Ab毒素具有较高的亲和力和特异性，结果表明，利用该方法能够由噬菌体随机肽库中高效捕获亲和序列，筛选到与活化 Cry2Ab毒素具有高亲和力的多肽，该序列可以模拟 Cry2Ab毒素的受体表位，为进一步研究 Cry2Ab毒素作用机制奠定了基础，并为今后田间抗性基因频率检测，以及毒素—受体作用机制研究工作提供更有力的技术支持。
英文摘要：The insecticidal Cry toxins produced by Bacillus thuringiensisare highly specific to different insects． Various proteins，such as cadherin，aminopeptidase-N (APN ) and alkaline phosphatase (ALP ) are characterized as potential Cry-receptors． However，little is known about the mode of Cry2Ab action，such as its receptors and binding sites． We used phage display to characterize the binding epitope of the Cry2Ab toxinin vitro． A two peptide sequence was identified after four-rounds of screening． ELISA analysis showed that activated Cry2Ab toxin could bind these two peptides with highaffinity． The results indicate that employing this method can efficiently screen out target peptides with high affinity andspecificity．The method also provides a valuable platform to discover the mode of other Bt toxins．