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目的:构建含有HIV-1负调控因子(Nef)基因的重组慢病毒表达载体,并检测Nef蛋白在人胚肾293T细胞、体腔渗出性淋巴瘤细胞BCBL-1和人血管内皮细胞EA.hy926中的表达情况及其对BCBL-1和EA.hy926细胞增殖的影响。方法:从本实验室已构建好的含Nef基因的重组真核表达质粒pCI-neo-Nef中扩增出Nef基因,插入到慢病毒载体pHAGE-CMV-MCS-Izs-Green中构建成pHAGE-Nef,利用脂质体将其与包装质粒psPAX2和包膜质粒pMD2.G共转染293T细胞。荧光显微镜下观察293T细胞中绿色荧光蛋白(GFP)的表达并收集培养上清,此上清经0.45μm滤器过滤后即获得慢病毒悬液。梯度稀释法测定慢病毒滴度。将重组慢病毒分别感染293T、BCBL-1和EA.hy926细胞,用Western blot检测Nef蛋白的表达。通过细胞增殖实验检测Nef蛋白对BCBL-1和EA.hy926细胞增殖的影响。结果:限制性内切酶鉴定和核酸序列测序证实成功构建了含HIV-1 Nef基因的重组慢病毒表达载体,病毒滴度为1×107 TU/ml。以重组慢病毒分别感染293T、BCBL-1和EA.hy926细胞,均能检测到Nef蛋白的表达,且Nef蛋白能够抑制BCBL-1和EA.hy926细胞的增殖作用。结论:成功构建了含HIV-1 Nef基因的慢病毒表达载体,获得的慢病毒不仅能够有效感染293T、BCBL-1和EA.hy926细胞,而且能够介导Nef蛋白在这些细胞中表达。重组慢病毒载体介导的Nef蛋白能够抑制BCBL-1和EA.hy926细胞的增殖。
OBJECTIVE: To construct a recombinant lentiviral vector containing the negative regulatory factor (Nef) gene of HIV-1 and to detect the expression of Nef protein in 293T cells of human embryonic kidney, BCBL-1 of exudative lymphoma cells and EA.hy926 And its effect on the proliferation of BCBL-1 and EA.hy926 cells. Methods: The Nef gene was amplified from the recombinant eukaryotic expression plasmid pCI-neo-Nef containing Nef gene in our laboratory and inserted into the lentiviral vector pHAGE-CMV-MCS-Izs-Green. Nef and 293T cells were co-transfected with packaging plasmid psPAX2 and envelope plasmid pMD2.G using liposomes. The expression of green fluorescent protein (GFP) in 293T cells was observed under a fluorescence microscope and the culture supernatant was collected. The supernatant was filtered through a 0.45 μm filter to obtain a lentivirus suspension. Gradient dilution method for determination of lentivirus titer. 293T, BCBL-1 and EA.hy926 cells were respectively infected with the recombinant lentivirus and Nef protein was detected by Western blot. The effect of Nef protein on the proliferation of BCBL-1 and EA.hy926 cells was detected by cell proliferation assay. Results: The restriction endonucleases and DNA sequencing confirmed that the recombinant lentiviral vector containing HIV-1 Nef gene was successfully constructed. The virus titer was 1 × 107 TU / ml. Nef protein was detected in 293T, BCBL-1 and EA.hy926 cells by recombinant lentivirus, and Nef protein could inhibit the proliferation of BCBL-1 and EA.hy926 cells. CONCLUSION: The lentiviral expression vector containing HIV-1 Nef gene was successfully constructed. The lentivirus obtained can not only effectively infect 293T, BCBL-1 and EA.hy926 cells, but also mediate the expression of Nef protein in these cells. The recombinant lentiviral vector-mediated Nef protein was able to inhibit the proliferation of BCBL-1 and EA.hy926 cells.