结核分枝杆菌Hsp16.3的表达、纯化和鉴定  

Expression,purification and identification of Hsp16.3 protein from Mycobacterium tuberculosis

作　　者：张廷芬[1] 师长宏[1] 朱德生[1] 张海[1] 赵勇[1] 白冰[1] 

机构地区：[1]第四军医大学实验动物研究中心,西安710032

出　　处：《中国人兽共患病学报》2007年第10期964-967,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助项目(No30670116)

摘　　要：目的克隆结核分枝杆菌hspx(acr、Rv2031c)基因,扩增后测序,并在大肠杆菌中表达获得基因重组蛋白Hsp16.3(Heat Shock Protein16.3)。方法用PCR方法从结核分枝杆菌H37Rv基因组扩增出hspx基因片段,连接进入pTA2载体中,序列测定正确后,将其亚克隆到表达载体pProEX HTb并在大肠杆菌DH5α中表达,表达蛋白SDS-PAGE分析后,分别与6×His mAb、16k Da mAb和结核病人血清进行Western-blot,最后用Ni-NTA进行亲和层析纯化蛋白。结果获得结核分枝杆菌Hsp16.3蛋白基因,经序列测定与GenBank公布的序列完全一致;表达蛋白经SDS-PAGE分析,相对分子质量与文献报道一致;Western-blot结果显示,在相对分子量约16kDa处有与6×His mAb和鼠抗16k Da mAb的特异性结合带,而与结核病人血清杂交没有出现结合带。表达产物为包涵体,通过Ni-NTA纯化系统,可得到纯化的目的蛋白。结论成功地表达、纯化和鉴定了Hsp16.3蛋白,它有可能作为新型结核疫苗的靶抗原。The hspx（acr,Rv2031c）gene Of Mycobacterium tuberculosis H37Rv was firstly amplified by PCR from gehome of M. tuberculpsis H37RV strain and then cloned into plasmid pTA2. After sequence, the hspx gene segment digested with BamH Ⅰ and Hind Ⅲ was subcloned to the expression vector pProEX HTb and expressed in E. coli DHSa. The expressed protein was identified by SDS-PAGE analysis and Western blot analysis with monoclonal antibody against 6 × His and 16 kDa protein and sera of tuberculous patients,and the recombinant 6 × His fused protein was purified by Ni-NTA purification system. It was demonstrated that the size of the PCR product was 435 bps and this sequence was identical to that of the hspx gene in GenBank. As demonstrated by SDS-PAGE analysis the relative molecular mass of this protein was consistent with that was previously reported. In Western blot analysis,a specific binding band of this protein with 6 × His mAb and mouse anti-16 kDa mAb could be detected at the corresponding site with relative molecular mass of 16 kDa,but no binding band was found with sera of patients with tuberculosis. It is evident that the Hsp16.3 protein was successfully expressed and purified,and it may be used as the target antigen for the novel tuberculous vaccine.

关 键 词：结核分枝杆菌 Hsp163 休眠 

分 类 号：R378.9[医药卫生—病原生物学]