苏云金芽孢杆菌以色列亚种毒性蛋白基因的克隆及其原核表达  

Gene cloning and prokaryotic expression of insecticidal crystal proteins from Bacillus thuringiensis subsp.Israelensis

作　　者：唐建霞 赵松[1] 戴洋[1] 顾晓红[1] 刘琴[3] 徐健[3] 管晓虹[2] 朱荫昌[1] 

机构地区：[1]江苏省血吸虫病防治研究所、卫生部寄生虫病预防与控制技术重点实验室、江苏省寄生虫分子生物学重点实验室、江苏省寄生虫病重点学科,无锡214064 [2]南京医科大学卫生部抗体技术重实验室 [3]江苏省里下河地区农业科学研究所

出　　处：《中国血吸虫病防治杂志》2009年第2期119-123,共5页Chinese Journal of Schistosomiasis Control

基　　金：江苏省卫生厅重大项目(K200514);江苏省科技计划项目(BS2005014)

摘　　要：目的从苏云金芽孢杆菌(Bacillus thuringiensis,Bti)以色列亚种中获得cry4B、cry11A和cyt1A3种主要杀蚊幼虫蛋白的基因,并进行克隆和原核表达。方法根据GenBank上3种基因的已知序列设计合成引物,应用PCR技术扩增目的基因,克隆入pUC19质粒,阳性克隆质粒经酶切和测序鉴定。将测序鉴定的3种蛋白基因克隆入原核表达载体(pET32c)进行原核表达。结果PCR扩增获得特异性的基因片段,重组质粒经酶切后获得预计大小的基因片段,并测序鉴定。序列结果与已知序列一致。原核表达载体经ITPG诱导,获得预计大小的目的蛋白。结论成功克隆了苏云金芽孢杆菌以色列亚种的3个主要杀蚊幼虫蛋白的基因,并进行原核表达。Objective To obtain insecticidal crystal proteins from Bacillus thuringiensis subsp, lsraelensis including cry4B cry11A and cyt1A and evaluate the prokaryotic expression. Methods Specific primers were designed according to the sequences published on GenBank and used for amplification of the three insecticidal crystal proteins from Bacillus thuringiensis subsp. Israelensis through PCR technique. The genes were sub-cloned into pUC19 plasmid and identified by restriction endonulease digestion analysis and DNA sequencing. After that these genes were sub-cloned into pET32c（ ＋ ） for expression. Results The full length sequences of cry4B cryl 1A and cytlA were obtained successfully. The recombinant proteins were cloned and expressed. Conclusions The insecticidal crystal proteins from Bacillus thuringiensis subsp, lsraelensis are successfully cloned and these recombinant proteins are also successfully expressed.

关 键 词：苏云金芽孢杆菌以色列亚种 基因克隆 原核表达 

分 类 号：R384.1[医药卫生—医学寄生虫学]