结核杆菌pIHSP65真核表达载体的构建和表达

目的构建结核杆菌热休克蛋白65KD(HSP65)基因真核表达载体pIHSP65,并在Hela细胞中表达。方法从结核杆菌H37Rv株的基因组中扩增HSP65基因,克隆到双顺反子真核表达载体pIRES的多克隆位点A中,构建pIHSP65真核表达载体,采用阳离子多聚体将其转染到Hela细胞中,用免疫组化染色法检测HSP65基因的表达。结果所克隆的HSP65基因片段经测序完全正确。重组质粒转染Hela细胞后,用免疫组化染色法检测有HSP65蛋白的表达。结论成功构建了结核杆菌pIHSP65真核表达载体,并在Hela细胞中有效表达,为构建双抗原双顺反子真核表达质粒,以及在整体动物水平的实验研究奠定了基础。

Construction and expression of eukaryotic expression vector pIHSP65 of mycobacterium tuberculosis

Objective To construct the eukaryotic expression vector based on HSP65 gene of mycobacterium tuberculosis.Methods HSP65 gene was cloned from genome of mycobacterium tuberculosis H37Rv strain by PCR,and inserted into multiple cloning site A's corresponding sites of dicistronic eukaryotic expression vector pIRES to construct eukaryotic expression vector pIHSP65,then transfected into Hela cell by using polycation.Finally,the expression of HSP65 protein was detected by immunochemical staining.Results The length and sequence of the cloned HSP65 segment were correct.The positive expression of HSP65 in Hela cell,which had been transfection with recombinant plasmid,was identified by immunohistochemical method.Conclusion The successful construction of eukaryotic expression vector pIHSP65 and effective expression in Hela cells,lays the foundation for constructing two-antigens dicistronic eukaryotic expression plasmid and further study in animal.