IRF5基因慢病毒表达载体的构建及其在巨噬细胞RAW264.7中的表达

目的: 构建干扰素凋节因子5(IRF5)慢病毒表达载体LV11-cmv-neo-IRF5并转染巨噬细胞RAW264.7,观察IRF5巨噬细胞RAW264.7中的表达。方法: 采用基因重组技术,将PCR技术钓取的目的基因IRF5片段克隆至线性化慢病毒表达载体LV11中,重组载体经PCR、双酶切和测序鉴定构建成功后,采用脂质体转染技术将表达质粒和包装质粒共转染293FT细胞,获得携带IRF5基因的重组慢病毒;收集病毒上清液感染小鼠巨噬细胞RAW264.7,用G418将非浸染的细胞致死,获得纯净的浸染细胞,即RAW264.7-IRF5细胞(实验组),并将RAW264.7-NC细胞作为对照(对照组)。分别采用RT-qPCR和Western blotting法检测2组细胞中IRF5mRNA和蛋白的表达水平。结果: 重组慢病毒质粒LV11-cmv-neo-IRF5经PCR和酶切法鉴定构建成功;病毒上清液感染RAW264.7细胞并经G418筛选获得阳性细胞;RT-qPCR法检测,与对照组比较,实验组细胞中IRF5mRNA表达水平升高(P<0.001);Western blotting法检测,实验组细胞中IRF5蛋白的表达水平明显高于对照组。结论: 成功构建携带IRF5基因的慢病毒表达载体LV11-cmv-neo-IRF5,IRF5基因在巨噬细胞RAW264.7中稳定表达。

Construction of IRF5 gene lentiviral vector and its expression in macrophages RAW264.7

Objective: To construct interferon regulatory factor 5(IRF5) gene lentiviral vector LV11-cmv-neo-IRF5 and transfect it into macrophages RAW264.7, and to observe its expression in RAW264.7 cells. Methods: The target gene IRF5 gragment was fished by PCR and cloned into linearized lentiviral vector LV11 by using DNA recombinant technique. The recombinant vector was identified by PCR, double enzyme digestion,and sequencing and the 293T cells were transfected with the lipofectin reagent for lentiviral particles packaged with expression and packing plasmids. The supernatant of lentiviral was collected and infected into the macrophages RAW264.7. After screening the cells, which were not infected with G418, the positive infected cells(RAW264.7-IRF5)were obtained (experiment group). Meanwhile, the RAW264.7-NC cells was regarded as controls(control group).The expression levels of IRF5 mRNA and protein in RAW264.7 cells in two groups were examined by RT-qPCR and Western blotting methods. Results: The PCR and enzyme digestion analysis results confirmed that the lentiviral vector LV11-cmv-neo-IRF5 was successfully constructed. The RAW264.7 cells were infected with virus supernatant and the positive cells were obtained by G418 screening.The RT-qPCR results showed that compared with control group, the expression level of IRF5 mRNA in the RAW264.7 cells in experiment group was increased (P<0.01);the Western blotting results showed that compared with control group,the IRF5 protein epression level in the RAW264.7 cells in experiment group was increased. Conclusion: The lentiviral vector LV11-cmv-neo-IRF5 containing IRF5 gene is successfully constructed,and the IRF5 gene can express stably in the macrophages RAW264.7.