中文摘要：

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（resuscitation-promoting factor-interacting protein A,RipA）,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（Isopropyl β-D-1-thiogalactopyranoside IPTG）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,分光光度法测定细菌的A600,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量为52kDa的目的蛋白,Western-blot结果显示该蛋白与6×His单抗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。

英文摘要：

Objective：To clone and express resuscitation-promoting factor-interacting protein A （RipA） of M.tuberculosis （MTB） in E.coli DH5a, and observe the resuscitation activity of purified RipA protein on the M.smegmatis.Methords：The Rv1477 gene encod-ing RipA protein was amplified by polymerase chain reaction （PCR）from genome of MTB H37Rv strain, and inserted into prokaryotic expression vector pPro-EX HTa.The recombinant plasmids were transformed into E.coli DH5, and induced with IPTG.The fusion protein was purified by Ni＋-NTA purification system with denative condition.The purified RipA protein was added into the 7H9 media culturing M.smegmatis, and the resuscitation activity of RipA protein was measured with spectrophotometry at a wave length of A600 nm.Results：Sequence of Rv1477 gene amplified by PCR was identical with that of Genbank reported.The positive recombinant clone was identified by restriction enzyme digestion analysis.The recombinant plasmid expressed fusion protein of RipA with relative molecular mass（Mr）of 52KDa, which was confirmed by western-blot analysis with specific monoclonal antibody against 6×His Ab.Fussed protein could be purified by Ni＋-NTA purification system and resuscitate the dormant M.smegmatis remarkably at 100pM concentrations in the 7H9 media.Conclusion：RipA protein of MTB was successfully expressed in E.Coli DH5, which could promote the growth of M.smegmatis significantly.