苏云金杆菌vip3 A基因的克隆、表达及杀虫活性分析  

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

作　　者：陈建武[1] 唐丽霞[1] 汤慕瑾[1] 师永霞[1] 庞义[1] 

机构地区：[1]中山大学生物防治国家重点实验室,广州510275

出　　处：《生物工程学报》2002年第6期687-692,共6页Chinese Journal of Biotechnology

基　　金：国家转基因植物专项基金资助项目 (No .J0 0 A 0 0 3);国家 86 3计划 (No.2 0 0 1AA2 14 0 11);广东省自然科学基金项目;中山大学研究团队项目~~

摘　　要：用全长PCR方法从野生型苏云金杆菌 (Bacillusthuringiensis,Bt)菌株S184中克隆了 2 3kb大小的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 LIPTG诱导后可表达 89kD大小的Vip3A S184蛋白 ,并得到Westernblot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 (Spodopteraexigua)、斜纹夜蛾 (S .litura)和棉铃虫 (Helicoverpaarmigera)等 3种害虫的初孵幼虫进行生物测定 ,结果表明 ,Vip3AThe 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.

关 键 词：苏云金杆菌 vip34基因 基因克隆 表达 杀虫活性分析 抗体 

分 类 号：Q78[生物学—分子生物学]