棉铃虫颗粒体病毒增效因子功能域和作用机理的研究

We obtained the enhancin gene from Helicoverpa armigera by PCR method and expressed this gene in E.coli. The plasmids pET-30a-Ben and pET-30a-Den which included 1.7kb and 2.2 kb fragments of 5'-terminal of HaGV enhancing gene were obtained by cutting recombinant plasmid pET-30a-En with Bal I and Dra I respectively. Two fragments were expressed in E.coli successfully and the products were named Ben and Den respectively. The enhancement, which Ben and Den enhance the infectivity of HaNPV and Bt in 3rd larvae of Helicoverpa armigera was studied. The results indicated that there was increase of the mortality of 10.5-26.5% and the LD50 decrease of 0.9d causes by adding Ben, while Den could increase the mortality by 10.2-33.0% and decrease the LD50 by 0.2-1.9d. The preliminary bioassay on Bt against Helicoverpa armigera indicated the recombinant enhancing could increase the mortality of larvae by 20.7-35.4%, Ben by 16.7-31.5%, Ben by 11.7-27.4%. Successfully expression using lactose in place of IPTG will be a good suggestion for the large scale production.

以棉铃虫颗粒体病毒为材料，从包涵体中分离增效因子及其降解产物，活性测定确定功能多肽，PCR扩增得到功能多肽的DNA序列，原核或真核体系表达功能多肽，活性检测确定增效因子的功能域。增效因子的全长基因经缺失和突变以验证结论，提出可能的增效机理，为在生产上应用此蛋白及探索新的更有效的抗虫策略奠定理论基础。