结核杆菌H37Rv株重组PstS1蛋白的表达纯化及其免疫反应性分析  

Study on expression,purification and immunoreactivity of mycobacteriuml tuberculosis recombinant PstS1

作　　者：石君帆[1] 宋广忠[1] 泮结超[1] 漏磊君[1] 李召东[2] 

机构地区：[1]浙江省医学科学院,浙江杭州310013 [2]杭州市红十字会医院

出　　处：《中国预防医学杂志》2011年第4期307-310,共4页Chinese Preventive Medicine

基　　金：浙江省科技厅专项公益技术攻关项目资助(2007F10033);浙江省医学科学院青年基金项目资助(A30810Q)

摘　　要：目的表达、纯化结核杆菌H37Rv株重组PstS1(简称rPstS1)蛋白,为结核病血清学诊断价值研究及亚单位疫苗研制提供物质基础。方法重组质粒pET15b-PstS1转化大肠埃希菌表达菌株BL21(DE3)plysE,IPTG诱导rPstS1蛋白表达。经SDS-PAGE电泳和Western blot鉴定后,优化表达条件,用镍离子鳌合亲和层析柱纯化rPstS1蛋白,并用斑点金免疫渗滤法评价纯化蛋白的免疫反应性。结果成功构建了重组质粒pET15b-PstS1,相对分子质量为38×103的rPstS1蛋白约有30%以可溶性蛋白形式表达,经亲和层析后的rPstS1蛋白纯度为94.8%,有较好的免疫反应性。结论构建的重组质粒pET15b-PstS1能高效表达有免疫反应性的rPstS1蛋白,经亲和层析后可得到高纯度的纯化蛋白。Objective To provide the basis for subunit vaccine development and serodiagnosis of tuberculosis by expressing and purifying phosphate-specific transport system 1（PstS1） gene from mycobacterium tuberculosis in E.coli.Methods First of all,recombinant plasmid pET15b-PstS1 was transformed into E.coli BL21（DE3） plysE,and then the recombinant PstS1（rPstS1） protein was expressed in E.coli BL21（DE3） plysE by inducement of IPTG.After identification by SDS-PAGE electrophoresis and western blot,the expression condition of rPsts1 was optimized.Then rPstS1 was purified by nickel-chelate affinity chromatography.Furthermore,the immunoreactivity of rPsts1 was evaluated by Dot-immunogold filtration assay.Results A recombinant plasmid pET15b-PstS1 was successfully constructed and could stably express a 38kDa rPstS1.The purity of rPsts1 reached 94.8% after affinity chromatography.Moreover the rPstS1 was confirmed to have a good immunoreactivity.Conclusion The successfully constructed recombinant plasmid pET15b-PstS1 could highly express rPstS1 protein with a good immunoreactivity,and the high-purified rPstS1 could be obtained by affinity chromatography.

关 键 词：Psts1 亲和层析 亚细胞定位 结核 

分 类 号：R392[医药卫生—免疫学]