结核分枝杆菌复苏因子Rv2450c基因的克隆、表达和纯化

Cloning and expression of M. tuberculosis resuscitation promoting factor Rv2450c

目的对结核分枝杆菌复苏因子Rv2450c基因克隆、并测序正确后进行融合蛋白表达、纯化。方法通过PCR扩增结核分枝杆菌H37Rv复苏因子Rv2450c基因,酶切,克隆至PGEM-TEasy质粒,测序确定插入片段的正确性,再克隆至表达载体pET30a中,并转化至BL21（DE3）中。经PCR鉴定阳性的菌液通过IPTG诱导,表达氨基端带6个连续组氨酸残基的Rv2450c蛋白,并纯化。结果获得了结核分枝杆菌复苏因子Rv2450c基因,构建了重组表达质粒,得到融合了6个连续组氨酸残基的Rv2450c蛋白,纯度〉90%。结论应用基因重组表达技术表达结核杆菌复苏因子蛋白,可为下一步摸索临床标本结核分枝杆菌复苏培养及复苏基因的功能研究打下一个良好的基础。

Objective To clone the gene of M. tuberculosis resuscitation promoting factor （Rpf） Rv2450 c,and after obstaining the right sequence, to study the expression and purification of the melting protein. Methods M. tuberculosis Rpf Rv2450c gene was amplified by PCR. Clone the gene into expression vector pET30a was cloned. The protein was expressed by IPTG induction. Results M. tuberculosis Rpf protein obtained was 〉90% purity. Conclusions The basis for further functional study and the resuscitable culture for M. tuberculosis is laid.