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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 the genes of cry4B, cry11A and cyt1A three major mosquito larvae from Bacillus thuringiensis (Bti) Israel, and to clone and prokaryote them. Methods The primers were designed according to the known sequences of three genes in GenBank. The target gene was amplified by PCR and cloned into pUC19 plasmid. The positive clones were identified by restriction enzyme digestion and sequencing. The three proteins identified by sequencing were cloned into prokaryotic expression vector (pET32c) for prokaryotic expression. Results Specific gene fragments were obtained by PCR amplification. The recombinant plasmids were digested and sequenced to obtain the gene fragments of the expected size. Sequence results are consistent with known sequences. The prokaryotic expression vector is induced by ITPG to obtain the target protein of the expected size. Conclusion The genes of three major mosquito larvae of Bacillus thuringiensis subsp. Pedatis were successfully cloned and prokaryotic expressed.