苏云金杆菌以色列亚种cryIVB基因在E.coli中的高表达

High Level Expression of cry IVB Gene of Bacillus thuringinesis var. israelensis in E.coli

韭菜迟眼蕈蚊（Ｂｒａｄｙｓｉａｏｄｏｒｉｐｈａｇａ）幼虫以取食根部危害韭菜的生长．为了研究有效的生防制剂，采用ＤＮＡ聚合酶链式反应方法，从苏云金杆菌以色列亚种（Ｂｔｉ）得到３．５ｋｂ的开放阅读框架（ＯＲＦ），其包括Ｂｔｉ杀虫蛋白基因ｃｒｙＩＶＢ的结构基因和部分上游启动区．将结构基因亚克隆到高表达载体ｐＱＥ５２中，构建了高表达质粒ｐＱＥ５２Ｂ．在Ｅ．ｃｏｌｉＳＧ１３００９（ｐＲＥＰ４）中，经ＩＰＴＧ诱导，１３０ｋＤ的蛋白表达量约占细胞总蛋白的７．９％，其对韭菜迟眼蕈蚊三龄幼虫具有较强的毒杀作用．为进一步利用ｃｒｙＩＶＢ基因。

Bradysia odoriphaga threatened the growth of Chinese chives with larvae root feeding activity.To study the engineered bacterial strain against B.odoriphaga ,a 3 5 kb open reading frame(ORF) including the cryIVB gene was obtained by PCR from B.thuringiensis var. israelensis. This ORF contains whole structural gene and partial upstream promoting region of cryIVB gene.Then the structural gene,without its start codon,was inserted into an E.coli expression vector,pQE52,and a new plasmid,pQE52B,was constructed.When transformed into E.coli SG13009(pREP4) and induced by IPTG,the cryIVB expressed 130 kD insecticidal protein at high level,representing about 7 9% of the total protein content in the E.coli cells,which can be checked with SDS PAGE.Bioassay revealed that the ICP was highly toxic to the third instar larvae of B.odoriphaga .This results lay a foundation of the construction of engineered bacterial strain.