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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。
Objective To transfect the HIV-1 Nef gene into THP-1 cells to obtain the cell clone stably expressing Nef protein, which lays the foundation for the study on the effect of Nef on macrophage biological activity. Methods THP-1 cells were transfected with plasmids pcDNA3.1 (+) - Nef and pcDNA3.1 (+ (negative), and the expression of THP-1 was detected by reverse transcriptase-polymerase chain reaction Methods The expression and localization of the target protein in eukaryotic cells were detected. The fluorescent reporter gene was transfected into THP-1 Nef and THP-13.1 cells by co-transfection method, and the biological activity of Nef protein was evaluated by measuring the fluorescence (RLU) value. Results The transfected cells were screened by G418 to obtain THP-1 cell line stably expressing Nef. The results of RT-PCR and Western blot showed that Nef was successfully expressed in eukaryotic cells. The results of immunofluorescence showed that THP-1-Nef cells expressed Nef protein is mainly located in the cytoplasm. The RLU values of THP-1-Nef and THP-1-3.1 cells transfected with HIV-1 LTR-Luc and NFκB-Luc fluorescence reporter were detected by fluorescence microplate reader. Conclusion The THP-1-Nef cells stably expressing cell line was successfully established and its biological activity was tested, which laid the foundation for further study of its mechanism of action.