PPE68/Ipr1真核共表达质粒的构建及鉴定  

Construction and Identification of Eukaryotic Coexpression Vector for PPE68/Ipr1

作　　者：靳志栋[1,2] 张丹[1,2] 何永林[1,2] 徐蕾[1,2] 佘茜[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,重庆400016 [2]重庆医科大学神经科学研究中心,400016

出　　处：《中国生物制品学杂志》2011年第4期410-413,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30771922;30901280);重庆市自然科学基金(2009BB5275)

摘　　要：目的构建结核分枝杆菌PPE68基因和小鼠胞内病原体抗性基因1(Intracellular pathogen resistance 1,Ipr1)的真核共表达质粒,并在小鼠巨噬细胞RAW264.7中表达。方法将结核分枝杆菌PPE68基因和小鼠Ipr1基因分别亚克隆至含多启动子的共表达载体pBudCE4.1中,构建真核共表达质粒pBud68-Ipr1,转染RAW264.7细胞,通过RT-PCR及Western blot法检测PPE68和Ipr1基因的转录和表达。结果重组表达质粒pBud68-Ipr1经PCR、双酶切及测序证实,插入的PPE68和Ipr1基因片段序列正确;质粒转染RAW264.7细胞后,PPE68和Ipr1基因进行了转录和表达,基因编码产物相对分子质量分别为37 000和50 000。结论已成功构建了真核共表达质粒pBud68-Ipr1,并在RAW264.7细胞中获得表达,为PPE68/Ipr1重组BCG的构建及其免疫保护作用的进一步研究奠定了基础。Objective To construct a eukaryotic vector for coexpression of PPE68 gene of Mycobacterium tuberculosis and intracellular pathogen resistance 1（Ipr1） gene of mice,and express in murine macrophage RAW264.7 cells.Methods PPE68 and Ipr1 genes were subcloned into coexpression vector pBudCE4.1 containing multiple promoters,and the constructed recombinant plasmid pBud68-Ipr1 was transfected to RAW264.7 cells.The transcriptions of PPE68 and Ipr1 genes were determined by RT-PCR,and their expressions by Western blot.Results PCR,restriction analysis and sequencing proved that both PPE68 and Ipr1 genes with correct sequences were inserted into recombinant plasmid pBud68-Ipr1.Both PPE68 and Ipr1 genes were transcribed and expressed in transfected RAW264.7 cells,and the relative molecular masses of expressed protein were 37 000 and 50 000 respectively.Conclusion Eukaryotic coexpression vector pBud68-Ipr1 was successfully constructed and expressed in RAW264.7 cells,which laid a foundation of construction and further study on the immune protection of recombinant BCG with PPE68/Ipr1.

关 键 词：结核病 PPE68 胞内病原体抗性基因1 巨噬细胞 

分 类 号：R378.911[医药卫生—病原生物学] Q78[医药卫生—基础医学]