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目的在大肠埃希氏菌中表达结核分枝杆菌复苏促进因子结合蛋白A蛋白(RipA),纯化后观察该蛋白对耻垢分枝杆菌的促生长作用。方法采用PCR方法 ,从结核分枝杆菌H37Rv的DNA中扩增出编码RipA蛋白的Rv1477基因,测序正确后克隆入原核表达载体pProEx HTa,构建重组表达质粒pProEx HTa-RipA。以重组质粒转化大肠杆菌DH5a,筛选阳性重组菌株,经异丙基硫代-β-D半乳糖苷诱导RipA表达,在变性条件下对目的蛋白进行亲和层析纯化。将纯化的RipA蛋白加入到耻垢分枝杆菌中,观察RipA蛋白的促生长作用。结果成功扩增了Rv1477基因,并克隆于表达载体pProEx HTa中,经酶切鉴定获得阳性克隆,经诱导,在大肠杆菌中表达出相对分子质量为52×103的目的蛋白,Western-blot结果显示该蛋白与6×HisAb有特异性的反应条带。采用Ni+-NTA柱可获得纯化的目的蛋白。100pM的RipA蛋白可显著促进耻垢分枝杆菌休眠菌的复苏和生长。结论 RipA蛋白在大肠杆菌中成功表达,并能有效促进耻垢分枝杆菌的生长,为MTB的快速诊断研究奠定了基础。
Objective To express Mycobacterium tuberculosis recovery promoting factor binding protein A protein (RipA) in Escherichia coli and observe the growth promoting effect of the protein on Mycobacterium smegmatis. Methods Rv1477 gene encoding RipA protein was amplified from the DNA of Mycobacterium tuberculosis H37Rv by PCR and cloned into the prokaryotic expression vector pProEx HTa to construct the recombinant expression plasmid pProEx HTa-RipA. The recombinant plasmid was transformed into E. coli DH5a, and the positive recombinant strains were screened. RipA expression was induced by isopropylthio-β-D-galactoside, and the target protein was purified by affinity chromatography under denaturing conditions. The purified RipA protein was added to Mycobacterium smegmatis to observe the growth promoting effect of RipA protein. Results The Rv1477 gene was successfully amplified and cloned into the expression vector pProEx HTa. The positive clones were identified by restriction enzyme digestion. The recombinant protein was expressed in E. coli with the molecular weight of 52 × 103. The results of Western-blot This protein has a specific reaction band with 6 × HisAb. Using Ni + -NTA column to obtain the purified target protein. 100pM RipA protein can significantly promote the recovery and growth of M. smegmatis resting bacteria. Conclusion RipA protein is successfully expressed in E. coli and can effectively promote the growth of M. smegmatis, which lays the foundation for the rapid diagnosis of MTB.