苏云金芽孢杆菌营养期杀虫蛋白基因的克隆、表达及杀虫活性分析

Cloning and expression product of vip3A gene fromBacillus thuringiensis and analysis of inseceicidal activity

选择本实验室分离的野生型苏云金芽孢杆菌菌株WY-197为出发菌株,用全长PCR方法从此菌株中克隆了2.3kb大小的vip3A基因.DNA序列比较发现所克隆的基因vip3A-197与已知的营养期杀虫蛋白基因存在很高的同源性.将基因vip3A-197亚克隆至原核表达载体pET33b构建了原核表达质粒pEVip,转化大肠杆菌BL21,转化子经IPTG诱导后可表达88kD大小的蛋白.该蛋白对甜菜夜蛾(Spodopteraexigua)和棉铃虫(Helicoverpaarmigera)的初孵幼虫进行生物测定,结果表明,营养期杀虫蛋白vip3A-197对夜蛾科害虫具有一定的杀虫活性.

<Abstrcat> The vip3A gene in a size of 2.3 kb from a wild Bacillus thuringiensis WY-197 was cloned and sequenced. Comparison of the amino acid sequence with those of reported VIPs revealed high similarity. Vip3A-197 gene was subcloned into prokaryotic expression vector pET33 b to form recombinant expression plasmid pEVip and was transfered into escherichia coli, BL21. Recombinant strain of E. coli BL21 induced by IPTG could produce 88 000 protein by SDS-PAGE. Bioassay also showed that vip3A-197 protein was toxic to Spodoptera exigua and Helicoverpa armigera larvae.