摘要
目的表达纯化藤黄微球菌（Micrococcus luteus）复活促进因子（Rpf）及结构域（domain）蛋白，研究其对结核分枝杆菌生长的影响。方法诱导表达含有藤黄微球菌Rpf及其结构域基因的融合表达载体pPro-EXHT—Rpf和pPro—EXHT-Rpf domain，在变性条件下对目的蛋白进行纯化。用不同浓度的纯化蛋白刺激不可培养状态结核分枝杆菌的生长。结果获得藤黄微球菌Rpf及其结构域融合蛋白的纯度分别为95％和93％，相对分子质量（Mr）大小分别为30×10^3和12×10^3，蛋白质含量分别为471mg／L和337ms／L。Western blot证实，获得的蛋白为我们所需要的目的蛋白。所获得的藤黄微球菌Rpf及其结构域融合蛋白能够促进不可培养状态的结核分枝杆菌的生长，而抗藤黄微球菌Rpf结构域的单克隆抗体可以明显抑制结核分枝杆菌的生长。结论表达的藤黄微球菌Rpf及其结构域融合蛋白可以促进结核分枝杆菌的生长，而且藤黄微球菌Rpf蛋白与Rpf结构域蛋白具有一致的生物活性。
Objective To purify Micrococcus luteus Rpf and Rpf domain fusion protein, and to investigate its effects on growth of Mycobacterium tuberculosis. Methods The recombinant plasmids pPro- EXHT-Rpf and pPro-EXHT-Rpf domain were expressed in E. coli DH5α and then purified under denaturing condition via Ni-NTA purification system and confirmed by Western blot. The biochemical property of the M. luteus Rpf and Rpf domain was analyzed by stimulating the resuscitation of M. tuberculosis H37Ra which were in non-cuhurable＇ condition. Results The Rpf and Rpf domain products achieved 95% and 93% pure respectively, and the molecular weight was 30 ×10^3 and 12×10^3 , the yield of purification was about 471 mg/L and 337 mg/L of the culture. The M. luteus Rpf and Rpf domain from the E. coli showed activity of stimulating the resuscitation of M. luteus and M. tuberculosis H37Ra in non-culturable＇ condition which could be inhibited by monoclonal antibodies of M. luteus Rpf domain remarkably. Conclusion It was demonstrated that the purification of Rpf and Rpf domain have high biological activity for further functional, pharmacological and clinical investigations, and M. luteus Rpf domain protein is fully active as M. luteus full-length Rpf.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2009年第6期503-506,共4页
Chinese Journal of Microbiology and Immunology
基金
国家863高技术研究发展计划资助项目（2007AA02Z473）
国家自然科学基金资助项目（30670116,30500432）
关键词
藤黄微球菌
结核分枝杆菌
复活促进因子
结构域
Micrococcus luteus
Mycobacterium tuberculosis
Resuscitation promoting factor
Domain
