Objective To construct an eukaryotic expression vector of bombyxin E in order to provide a basis for further study of the hypoglycemic effects in mammalian cell .Methods Bbx E cDNA was amplified by PCR .Plasmid PUC 57 which contain Bbx E cDNA was used as template .The fragment was digested and ligated to the eukaryotic expression vector PcDNA 3 .1 The ligation mixture was transformed into competent E .coli DH-5a cells .Transformants containing inserts were confirmed by restrictive digestion and DNA sequencing .Results The PCR product was about 300 bp .The recombinant expression vector was identified by restrictive digestion and DNA sequencing .Conclusion The recombinant E eukaryotic expression vector PcDNA 3 .1-BbxⅡ/His is constructed successful—ly.%目的 构建(BbxⅡ)基因真核表达载体,为研究家蚕素Ⅱ在哺乳动物细胞中的生物学作用奠定基础.方法 以含有BbxⅡcDNA 的PUC57质粒为模板,采用聚合酶链式反应(PCR)扩增BbxⅡcDNA片段.真核表达载体PcDNA3.1及PCR产物经双酶切后连接,转化大肠杆菌DH-5α感受态菌,获得重组载体PcDNA3.1-Bbx/His,进行酶切鉴定和测序鉴定.结果 PCR获得与预期大小一致约300 bp的特异性DNA,重组载体PcDNA3.1-BbxⅡ/His经双酶切及测序鉴定证实,BbxⅡ基因的cDNA片段正确插入真核表达载体中.结论 成功构建含有家蚕素基因Ⅱ的真核表达载体.
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