cry1Ac基因启动子表达Vip3Aa蛋白特性分析

苏云金芽胞杆菌cry基因启动子常用于构建蛋白表达载体。为探讨苏云金芽胞杆菌cry基因启动子指导Vip3Aa蛋白表达情况及杀虫活性,以p UC19载体为基础,运用重叠引物PCR方法构建Vip3Aa11表达载体,并与由T7启动子指导的Vip3Aa11表达蛋白杀虫活性、抗胰蛋白酶稳定性比较,初步探索发酵条件。结果表明,cry1Ac启动子与T7启动子均在上清液中表达大小为88 ku Vip3Aa11蛋白,对甜菜夜蛾、棉铃虫杀虫活性差异不显著,cry1Ac基因启动子在37℃、48 h更适合Vip3Aa11蛋白的表达,为vip基因表达、功能验证及杀虫机理等研究提供新思路。

Analysis on expressed Vip3Aa protein by cry1Ac promoter

cry promoter of Bacillus thuringiensis is commonly engineered to construct protein express vectors.In order to explore cry promoter of Bacillus thuringiensis that expresses Vip3Aa and analyzes the insecticidal activity,a vip3Aa11 gene was fused to cry1Ac promoter by overlap extension PCR,and then inserted into pUC19 vector,expressed in E.coli JM109.In this study,compared with Vip3Aa11 protein expressed under the promoter cry1Ac and T7 in insecticidal effect,resistances to trypsin and fermentation condition were analyzed,respectively.The results showed that both were expressed 88 ku protein in the supematant,the insecticidal activity of expressed protein under the promoter of cry1Ac and T7 had no significant difference to Spodoptera exigua and Helicoverpa Armigera,respectively.cry1Ac gene promoter was more suitable for Vip3Aa protein expression in 37 ℃ and 48 h.The results would provide a convenient way for vip gene expression,function verification and insecticidal mechanism etc.