【作者】 刘忠泉； 邢爱英； 贾红彦； 李自慧； 郑晓静； 曹庭明； 杜风娇； 杜博平； 古淑香； 张宗德；

【Author】 Zhongquan Liu,Aiying Xing,et al.Beijing Tuberculosis and Thoracic Tumor Research Institute,Beijing 101149,China

【机构】 北京市结核病胸部肿瘤研究所结核病分子生物学研究室；

【摘要】 目的构建结核分枝杆菌复苏因子E基因的真核表达质粒。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Westen blot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Westen blot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。结论我们成功的构建了复苏因子E真核表达系统。更多还原

【Abstract】 Objective To construct the eukaryotic expression plasmid of Resuscitation Promoting factor E of M.tuberculosis.Methods Amplification Resuscitation Promoting factor E gene by PCR.The fragment was cloned into PcDNA3.1(-) vector,after transfected to CHO cells with it,Expression protein of Resuscitation Promoting facto E.Detected expression of mRNA in eukaryotic cells by RT-PCR,collected and purified protein, detected target protein with SDS-PAGE analysis and Westen blot analysis,Identification of the immunogen of expression protein by lymphocyte proliferation assay(MTT).Results The successful construction of the eukaryotic expression plasmid of Resuscitation Promoting factor E of M.tuberculosis.confirmed the gene mRNA expression in eukaryotic cells by RT-PCR.Confirmed the target protein expression by SDS-PAGE analysis,and Westen blot analysis.By lymphocyte proliferation assay(cell counting kit-8,CCK-8 ),further confirmed that the expressed protein is immunogenic.Conclusion We successfully constructed the eukaryotic expression system of Resuscitation Promoting factor E.更多还原