含有alpha;-胰凝乳蛋白酶作用位点的Cry3A突变体的构建、表达、纯化以及杀虫活性分析.pdfVIP

中国农业科学 2011,44(7): 1384-1389 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2011.07.010 含有α-胰凝乳蛋白酶作用位点的Cry3A 突变体的构建、 表达、纯化以及杀虫活性分析 刘京国，赵晓萌，杨爱珍，师光禄 （北京农学院生物技术学院/农业部都市农业（北方）重点开放实验室，北京 102206） 摘要：【目的】构建含有α-胰凝乳蛋白酶作用位点的Cry3A 的突变体Cry3Am，以期提高Cry3A 的活化效率， 增强杀虫活性。【方法】利用重叠PCR 技术，构建Cry3A 杀虫蛋白的突变体Cry3Am，使之在原核细胞中表达纯化 突变体蛋白；将Cry3Am 与饲料混合后饲喂黄粉虫，用以评价Cry3Am 的杀虫活性。【结果】成功地在Cry3A 杀虫蛋 白结构域I 的α3 和α4 之间加上1 个α-胰凝乳蛋白酶的作用位点，并提取Cry3Am 到杀虫蛋白。体外酶切试验结 果表明，Cry3Am 很容易地被降解为55 kD 的杀虫蛋白。生测分析结果表明，Cry3Am 对黄粉虫的毒力是Cry3A 的2 倍以上。【结论】向Cry3A 中插入α-胰凝乳蛋白酶作用位点可以使突变体Cry3Am 很容易地被α-胰凝乳蛋白酶降 解成55 kD 的片段，并能提高Cry3A 的杀虫活性。 关键词：Cry3A 突变体；α-胰凝乳蛋白酶；表达纯化；杀虫活性分析 Construction, Expression, Purification and Insecticidal Activity Analysis of Cry3A Mutant with α-Chymotrypsin Site LIU Jing-guo, ZHAO Xiao-meng, YANG Ai-zhen, SHI Guang-lu (College of Biotechnology, Beijing University of Agriculture/Key Laboratory of Urban Agriculture (North), Ministry of Agriculture, Beijing 102206) Abstract: 【Objective 】The objective of this study is to construct a Cry3A mutant containing a α-chymotrypsin site, and to enhance its insecticidal activity. 【Method 】 The Cry3Am was constructed by overlap PCR and the Cry3Am protein was purified. Tenebrio molitor were fed with Cry3Am and rolled oats to evaluate the toxicity of Cry3Am. 【Result 】 The Cry3Am with a α- chymotrypsin site at the linker between α3 and α4 of Cry3A domain I was constructed successfully. In vitro digestion result showed that Cry3Am was easier to be digested to 55 kD fragment than Cry3A. In vivo bioassay result indicated that the toxicity of Cry3Am against T. molitor was two times stronger than that of Cry3A. 【Conclusion 】 The insertion of α-chymotrypsin site resulted i