结核分枝杆菌PPE68基因真核表达质粒的构建及鉴定

Construction and identification of eukaryotic expression plasmid pBudCE4.1-PPE68 of Mycobacterium tuberculosis

目的:构建结核分枝杆菌(Mycobacteria tuberculosis,Mtb)PPE68基因的真核表达质粒,观察PPE68基因在小鼠巨噬细胞株RAW264.7中的表达。方法:将MtbPPE68基因克隆亚至真核表达质粒pBudCE4.1中,构建真核表达质粒pBudCE4.1-PPE68。以真核表达质粒pBudCE4.1-PPE68体外转染小鼠巨噬细胞株RAW264.7细胞,通过RT-PCR及Western blot法分别在转录水平及翻译水平检测PPE68基因的表达。结果:重组表达质粒经双酶切所切下的片段大小与理论值相符,测序结果与Gen-bank注录的PPE68基因序列一致。重组质粒瞬时转染小鼠巨噬细胞株RAW264.7,用RT-PCR及Western blot法分别在转录水平及翻译水平检测PPE68基因的表达。结论:成功构建了PPE68基因的重组真核表达质粒pBudCE4.1-PPE68,并在小鼠巨噬细胞株RAW264.7中得到了成功表达。为对其免疫原性、免疫反应性及免疫保护作用的进一步研究奠定了基础。

Objective：To construct and identify eukaryotic expression plasmid pBudCE4.1-PPE68 and observe its expression in murine macrophage RAW264.7 cells.Methods：MTB PPE68 gene was subcloned into pBudCE4.1 to construct recombinant plasmid pBudCE4.1-PPE68.The recombinant plasmid was transiently transfected into RAW264.7 cells,and the expression of PPE68 gene was detected by RT-PCR and Western blot.Results：The accurate eukaryotic recombinant plasmid pBudCE4.1-PPE68 was identified by restrict endonuclease.The PPE68 gene expression product in RAW264.7 cells was detected by RT-PCR、Western blotting.Conclusion：The recombinant plasmid pBudCE4.1-PPE68 was successfully constructed.The protein can be expressed in murine macrophage RAW264.7.The present study provides an experimental basis for further study of the PPE68 protein of Mycobacterium tuberculosis.