缺失α1螺旋的Cry1Ac在大肠杆菌中的表达、纯化和活性分析

Expression,Purification and Insecticidal Activity Analysis of Cry1Ac without Helix α-1

[目的]对缺失α1螺旋的Cry1Ac在大肠杆菌中的表达、纯化和活性进行分析。[方法]以苏云金芽孢杆菌HD-1中的质粒为模板,扩增缺乏α螺旋的Cry1Ac基因,并将其与pET28a载体连接,构建重组质粒。重组质粒转化并经IPTG诱导后,用SDS-PAGE检测目的蛋白的表达。提取目标蛋白,并涂在棉铃虫饲料表面,检测其杀虫活性。[结果]Cry1Acdelα1基因在大肠杆菌中能正常表达目标蛋白,但该目标蛋白是以包涵体形式存在。生侧结果显示,在相同浓度下,活化的Cry1Ac对棉铃虫的致死率达75%以上,而Cry1Acdelα1只有10%左右。[结论]用大肠杆菌表达的Cry1Acdelα1对棉铃虫基本没有杀虫活性。

[Objective] This study aimed to analyze the expression,purification and insecticidal activity of Cry1Ac without helix α-1 of domain I in E.coli.[Method] Plasmid of B.thuringiensis HD-1 was used as the template for amplification of Cry1Ac gene without α1 helix,which was linked with pET28a vector to construct the recombinant plasmid.After transformation and IPTG induction,expression of target protein was detected by using SDS-PAGE method.Target protein was extracted and coated on the surface of feed for H.armigera to determine the insecticidal activity.[Result] Cry1Acdelα1 gene could normally express the target protein in E.coli.However,the target protein existed in the form of inclusion body.Results of bioassay analysis showed that when under the same concentration,fatality rate of activated Cry1Ac toxin had reached above 75%,while that of Cry1Acdelα1 was only about 10%.[Conclusion] E.coli-expressed Cry1Acdelα1 had no insecticidal activity against H.armigera.