结核分枝杆菌复苏因子E的真核表达及鉴定

Eukaryotic expression and identification of resuscitation promoting factor E of Mycobacterium tuberculosis

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PcDNA 3.1（一）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Western blot分析检测目的 蛋白的表达,淋巴细胞增殖实验（CCK-8）鉴定表达蛋白的免疫原性.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Western 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 Mycobacterium tuberculosis. Methods Resuscitation promoting factor E gene fragment of Mycobacterium tuberculosis was amplified by PCR. The fragment was cloned into PcDNA 3.1 （-）vector. After sequencing the recombinant plasmid, it was transfected into CHO cells to express resuscitation promoting factor E protein. The expression of cloned gene mRNA in eukaryotic cells was detected by RT-PCR. The protein was collected and purified, and the targeted protein expression was detected by SDS-PAGE analysis and Western blot analysis. The immunogenicity of protein was identified by lymphocyte proliferation assay （CCK-8）. Results The eukaryotic expression plasmid of resuscitation promoting factor E of Mycobacterium tuberculosis was successfully constructed. It was confirmed that the cloned gene mRNA was expressed in the eukaryotic cells,the targeted protein was successfully expressed in the eukaryotic cells, and the expressed protein had immunogenicity. Conclusions The eukaryotic expression system of resuscitation promoting factor E was successfully constructed.