藤黄微球菌复苏促进因子结构域及其突变体基因的克隆和表达及生物活性的测定  

Expression in E. coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants

作　　者：岳晨莉[1] 史皆然[1] 师长宏[2] 张海[2] 赵雷[1] 张廷芬[2] 赵勇[2] 席丽[2] 

机构地区：[1]第四军医大学西京医院呼吸科,西安710032 [2]第四军医大学实验动物中心

出　　处：《中华结核和呼吸杂志》2008年第10期761-765,共5页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：国家“863”高技术研究发展计划基金资助项目（2007AA02Z473）;国家自然科学基金资助项目（30670116）;陕西省自然科学基金资助项目（2006C218）

摘　　要：目的克隆、表达藤黄微球菌复苏促进因子（Rpf）结构域及其突变体基因，评价其生物学活性。方法采用PCR方法从藤黄微球菌基因组中扩增出Rpf结构域及其突变体基因E54A、E54K，用限制性内切酶消化后克隆入pMD-18T载体中，将测序正确的基因插入到pGEX-4T-1表达载体中，转化大肠杆菌DH5α，经异丙基-β—D硫代半乳糖（IPTG）诱导，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定表达蛋白，利用Rpf结构域单克隆抗体进行Western blot分析。用GS-4B亲和层析柱纯化蛋白，将纯化的蛋白加入到休眠的耻垢分枝杆菌中测定各自的生物活性。结果获得藤黄微球菌Rpf结构域及其E54K和E54A突变体基因，测序结果与美国基因序列数据库GenBank公布的序列完全相同，突变位点与设定一致；表达蛋白相对分子质量与文献报道一致；Western blot结果显示，在相对分子质量约32000处有与Rpf结构域单克隆抗体特异性结合带。通过GS-4B系统，得到纯化的GST融合蛋白，并证实Rpf结构域蛋白对休眠的耻垢分枝杆菌有一定的复苏和促生长作用，突变体E54K对耻垢分枝杆菌的生长有抑制作用。结论获得Rpf结构域及其2种突变体蛋白，明确其对耻垢休眠菌有复苏的作用。Objective To express Micrococcus luteus resuscitation promoting factor（Rpf） domain and its mutants in prokaryotic cells, and to investigate their bioactivity. Methods The gene of Rpf domain and its mutants（E54K, E54A） were amplified by polymerase chain reaction（PCR）from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5α. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain （mAb）. The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. Results The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. Conclusions Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

关 键 词：分枝杆菌 耻垢 微球菌 藤黄 Rpf结构域 突变体 生物学活性 

分 类 号：R686[医药卫生—骨科学]