结核分枝杆菌复苏因子E的真核表达及鉴定  

The Eukaryotic Expression and Identification of Resuscitation Promoting Factor E of Mycobacterium tuberculosis in CHO Cells

作　　者：刘忠泉[1] 邢爱英[1] 贾红彦[1] 李自慧[1] 郑晓静[1] 曾庭明 杜风娇[1] 杜博平 古淑香[1] 张宗德[1] 

机构地区：[1]北京市结核病胸部肿瘤研究所结核病分子生物学研究室

出　　处：《结核病与胸部肿瘤》2010年第3期168-173,共6页Tuberculosis and Thoracic Tumor

基　　金：基金项目：国家重大专项（2008ZX100-03-001）.

摘　　要：目的构建结核分枝杆菌复苏因子ENN的真核表达质粒。方法PCR扩增结核分枝杆菌复苏因子E基因片段。将片段克隆至PcDNA3．1（-）载体，重组质粒测序正确后，转染至CHO细胞中，表达复苏因子E蛋白质。RT—PCR检测克隆基因mRNA在真核细胞中的表达，收集并纯化表达的蛋白质，SDS—PAGE分析和Westenblot分析检测目的蛋白的表达，淋巴细胞增殖实验（CCK-8）鉴定表达蛋的免疫原性。结果成功构建了结核分枝杆菌复苏因子E基因的真核表达质粒。RT—PCR证实克隆基因mRNA在真核细胞中表达，SDS-PAGE分析和Westenblot分析均证实目的蛋白在真核细胞中成功表达，淋巴细胞增殖实验（CCK-8）进一步证实了表达蛋白具有免疫原性。结论我们成功地构建了复苏因子E真核表达系统。Objective To construct the eukaryotic expression plasmid of resuscitation promoting factor E of M. tuberculosis. Methods Gene fragment of resuscitation promoting factor E of M. tuberculosis was amplified by PCR. Then the fragment was cloned into PcDNA3.1 （-） vector, which was transfeeted to CHO cells with it and expressed protein of resuscitation promoting factor E after recombination plasmid was sequenced correctly. Expression of mRNA in eukaryotic cells was detected by RT-PCR, and expressed protein was collected and purified. Target protein was detected with SDS-PAGE analysis and Westen blot analysis. Immunogen of expressed protein was identified by lymphocyte proliferation assay （MTT）. Results Eukaryotic expression plasmid of resuscitation promoting factor E of M. tuberculosis was successfully constructed, the gene mRNA expression in eukaryotic cells confirmed by RT-PCR, and the target protein expression confirmed by SDS-PAGE analysis, and Westen blot analysis. Lymphocyte proliferation assay （cell counting kit-8, CCK-8 ） confirmed further that the expressed protein is immunogenic. Conclusion We successfully constructed the eukaryotic expression system of resuscitation promoting factor E.

关 键 词：真核表达 复苏因子E 中国仓鼠细胞 

分 类 号：R378.911[医药卫生—病原生物学]