HIV-1 Nef基因在THP-1细胞的表达和活性检测

目的将HIV-1 Nef基因转染THP-1细胞,获得稳定表达Nef蛋白的细胞克隆,为研究Nef对巨噬细胞生物学活性的影响奠定实验基础。方法将质粒pcDNA3.1(+)-Nef和pcDNA3.1(+()阴性对照)转染THP-1细胞,通过逆转录-聚合酶链反应(RT-PCR)、Western blot、细胞免疫荧光等方法检测目的蛋白在真核细胞内的表达及定位,采用共转染法将荧光报告基因转染THP-1Nef和THP-13.1细胞,通过测定荧光(RLU)值来评价Nef蛋白的生物学活性。结果转染细胞经G418筛选后获得稳定表达Nef的THP-1细胞株,RT-PCR及Western blot结果表明Nef在真核细胞中成功表达,细胞免疫荧光结果显示,THP-1-Nef细胞表达的Nef蛋白主要定位于细胞质中。荧光酶标仪检测转染了HIV-1LTR-Luc和NFκB-Luc荧光报告基因的THP-1-Nef和THP-1-3.1细胞的RLU值。结论成功建立了THP-1-Nef细胞稳定表达细胞株,检测了其生物学活性,为进一步研究其作用机制实验奠定基础。

Establishment of HIV-1 Nef stable expressed cell strain in THP-1 cells and the activity detection

To investigate the effects of HIV-1 negative regulate factor(Nef) gene on THP-1 cells,we established Nef stable expressing cells.Plasmids pcDNA 3.1(+)-Nef and pcDNA 3.1(+) were stably transfected into THP-1 cells,respectively.Then the expression and location of Nef protein were confirmed by RT-PCR,Western blotting and cell immunofluorescence.Fluorescent reporter gene was transfected into THP-1 Nef and THP-1 3.1 cells respectively by Co-transfection or evaluating the biological activity of Nef protein.Finall...