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目的构建结核杆菌Rv0901基因的真核表达质粒并进行表达,为研究Rv0901基因的功能提供材料。方法以结核杆菌基因组DNA为模板PCR扩增Rv0901基因序列,将Rv0901基因定向克隆到真核表达质粒pcDNA3.1(+)获得重组表达质粒,体外转染Cos7细胞,RT-PCR检测转染的情况,并提取转染细胞总蛋白和收集细胞培养液上清检测蛋白的表达,Western-blotting检测蛋白表达的特异性。结果成功扩增出Rv0901基因序列,成功构建重组真核表达质粒pcDNA3.1-Rv0901,重组质粒转染细胞后的RT-PCR能扩增出Rv0901基因序列,转染细胞总蛋白和细胞培养液上清均能特异表达Rv0901基因蛋白。结论成功构建Rv0901基因真核表达质粒并在真核细胞内进行了良好表达,为研究该基因功能打下了坚实基础。
Objective To construct and express the eukaryotic expression plasmid of Mycobacterium tuberculosis Rv0901 gene and provide material for studying the function of Rv0901 gene. Methods The gene sequence of Rv0901 was amplified by PCR using Mycobacterium tuberculosis genomic DNA as a template. The Rv0901 gene was cloned into the eukaryotic expression plasmid pcDNA3.1 (+) to obtain the recombinant plasmid. The transfection was performed in vitro to Cos7 cells. , And the total protein of the transfected cells was collected and the supernatant of the cell culture supernatant was collected to detect the protein expression. The specificity of the protein expression was detected by Western-blotting. Results The Rv0901 gene sequence was successfully amplified and the recombinant eukaryotic expression plasmid pcDNA3.1-Rv0901 was successfully constructed. The RT-PCR of the recombinant plasmid transfected with the recombinant plasmid can amplify the Rv0901 gene sequence, and the transfected cells' total protein and cell culture medium Clear all can express Rv0901 gene protein. Conclusion The eukaryotic expression plasmid of Rv0901 gene was successfully constructed and expressed well in eukaryotic cells, which laid a solid foundation for studying the function of this gene.