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

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.