结核分支杆菌重组蛋白rCFP-10的表达、纯化及免疫反应性分析  

Study on Expression, Purification and Immunoreactivity of Mycobacteriuml Tuberculosis Recombinant CFP-10

作　　者：宋广忠[1] 石君帆[1] 泮结超[1] 杨明瑾[1] 漏磊君[1] John T. Belisle 

机构地区：[1]浙江省医学科学院寄生虫病研究所,杭州310013 [2]美国科罗拉多大学大学

出　　处：《浙江省医学科学院学报》2009年第1期11-15,共5页

基　　金：基金项目：浙江省科技厅重大项目资助（NO.2007F10033）

摘　　要：目的表达、纯化结核分支杆菌H37Rv株重组蛋白rCFP—10，评价其免疫反应性，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pET23b—CFP-10转化E．coli表达菌株BL21（DE3）pLysE，IPTG诱导重组蛋白rCFP-10表达。经SDS—PAGE电泳和Westernblotting鉴定后，优化表达条件用镍离子鳌合亲和层析柱HisTrap^TMHP纯化重组蛋白，最后用斑点金免疫渗滤法初步评价纯化蛋白的免疫反应性。结果成功构建了重组质粒pET23b—CFP-10并且重组蛋白rCFP-10以可溶性形式高效表达，表达量约占菌体总蛋白的10％。经亲和层析后得到高纯度有免疫反应性的重组蛋白（纯度约为98．5％）。结论高纯度有免疫反应性的重组蛋白rGFP-10为新型亚单位疫苗的研发及其结核病血清学诊断价值研究奠定了基础。Objective To investigate the expression and purification of mycobacterium tuberculosis recombinant CFP-10, providing material for tuberculosis （TB） serodiagnosis and subunit vaccine research. Methods Recombinant plasmid pET23b-CFP-10 was transformed into E.coli BL21 （DE3）pLysE, and the recombinant protein rCFP-10 was expressed in E.eoli BL21（DE3）pLysE by induction with IPTG. After identification by SDS-PAGE electrophoresis and western blotting, the expression condition of rCFP-10 was optimized. Then rCFP-10 was purified by nickel-chelate affinity chromatography. Results A recombinant plasmid pET23b-CFP-10 had been successfully constructed and could stably express about a 10KDa rCFP-10, existed in the form of solution. SDS-PAGE analysis showed a high expression rate of rCFP-10 which constituted about 10% of the total bacterial proteins. The high level of purity of rCFP-10 （about 98.5%） had been obtained by affinity chromatography. Conclusion The high-purity rCFP-10 was obtained.h could provide an experimental basis for TB serodiagnosis and subunit vaccine research.

关 键 词：CFP-10 血清学诊断 亚单位疫苗 结核 

分 类 号：R742.1[医药卫生—神经病学与精神病学] R378.911[医药卫生—临床医学]