苏云金杆菌vip3A基因的克隆、表达及杀虫活性分析

用全长PCR方法从野生型苏云金杆菌（Bacillus thuringiensis ,Bt)菌株S184中克隆了2.3kb大 vip3A基因并进行了序列分析。将vip3A-S184基因插入表达载体pQE30构建了表达质粒pOTP，转化大肠杆菌M15，转化子经1mmol/L IPTG诱导后可表达89kD大小的Vip3A-S184蛋白，并得到Western blot证实。蛋白可溶性试验表明，目的蛋白中约有19％是可溶的，用透射电镜观察到大多数蛋白是以包涵体形式存在的。因此，可以在自然条件下进行目的蛋白的纯化和对家兔进行免疫制备多克隆抗体，用于苏云金杆菌Vip3A蛋白表达的检测。利用IPTG进行诱导培养的菌液对甜菜夜蛾（Spodoptera exigua），斜纹夜蛾（S.litura)和棉铃虫（Helicoverpa armigera)等3种害虫的初孵幼虫进行生物测定，结果表明，Vip3A-S184蛋白对夜蛾科害虫具有较高的杀虫活性。

Cloning and Expression Product of vip3A Gene fr om Bacillus thuringiensis and Analysis of Inseceicidal Activity

The vip3A gene in a size of 2.3kb amplified from wild-type Bacillus thuringiensis strain S184 by PCR was cloned into pGEM-T Easy vector and its sequence was analysized by DNASTAR.The plasmid pOTP was constructed by inserting vip3A-S184 gene into the expression vector pQE30 and then was transformed into E.coli M15.E.coli M15 cells harbouring the plasmid pOTP were induced with 1mmol/L IPTG to express 89kD protein which was confirmed to be Vip3A-S184 by Western blot.Experiments showed that about 19% of Vip3A-S184 proteins were soluble,and others were insoluble proteins and formed inclusion bodies observed by transmission electron microscopy(TEM).The target protein was purified under the native condition and the polyclonal antibody was prepared by immunizing rabbits.The polyclonal antibody was used to detect Vip3A proteins expressed in Bacillus thuringiensis.Bioassay showed that Vip3A-S184 showed a high toxicity against 3 tested insect larvae including Spodoptera exigua,Spodoptera litura and Helicoverpa armigera.