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

作　　者：靳志栋[1,2] 何永林[1,2] 徐蕾[1,2] 佘茜[1,2] 张丹[1,2] 张壮苗[1,2] 杨春[1,2] 

机构地区：[1]重庆医科大学病原生物学教研室 [2]重庆医科大学神经科学研究中心重庆市重点实验室,重庆400016

出　　处：《重庆医科大学学报》2011年第8期907-910,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:30771922、30901280)

摘　　要：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.

关 键 词：MTB PPE68 巨噬细胞 

分 类 号：R337[医药卫生—人体生理学]