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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 eukaryotic co-expression plasmid of Mycobacterium tuberculosis PPE68 gene and mouse intracellular pathogen resistance 1 (Ipr1) and express in mouse macrophage RAW264.7 cells. Methods Mycobacterium tuberculosis PPE68 gene and mouse Ipr1 gene were subcloned into the co-expression vector pBudCE4.1 containing multiple promoters, respectively. The eukaryotic co-expression plasmid pBud68-Ipr1 was constructed and transfected into RAW264.7 cells. PCR and Western blot assay PPE68 and Ipr1 gene transcription and expression. Results The recombinant plasmid pBud68-Ipr1 was confirmed by PCR, double enzyme digestion and sequencing. The sequence of inserted PPE68 and Ipr1 gene fragments was correct. After transfected into RAW264.7 cells, the PPE68 and Ipr1 genes were transcribed and expressed. The molecular mass is 37 000 and 50 000 respectively. Conclusion The eukaryotic co-expression plasmid pBud68-Ipr1 was successfully constructed and expressed in RAW264.7 cells, which laid the foundation for the further study on the construction of recombinant BCG of PPE68 / Ipr1 and its immunoprotection.