【摘要】 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。更多还原

【Abstract】 Objective: To construct the recombinant lentivirus containing HIV-1 Nef gene and detect the protein expression in its target cell lines,such as 293T,BCBL-1 and EA.hy926,and explore the effect of Nef on cell proliferation.Methods:The fragment of Nef gene from expression vector pCI-neo-Nef was cloned into the lentivirus vector pHAGE-CMV-MCS-Izs-Green,then the recombinant vector pHAGE-Nef,packaging vector psPAX2 and envelope vector pMD2.G were cotransfected into the 293T cells.Culture media were harvested and filtered through a 0.45 μm filter to remove the cells.The viral titer was checked by observing the expression of green fluorescent protein(GFP).After infection of the recombinant lentivirus,the protein expression of Nef in 293T,BCBL-1 and EA.hy926 cells were detected by Western blot.The effect of Nef on cell proliferation was investigated by Cell Counting Kit-8 assay.Results:The recombinant lentivirus vector carrying HIV-1 Nef was constructed successfully.The viral titer was 1×107 TU/ml.293T,BCBL-1 and EA.hy926 cells could be efficiently infected by it.The expression of Nef in these cell lines could be detected by Western blot.In addition,Nef protein could inhibit the proliferation of BCBL-1 and EA.hy926 cells.Conclusion:Recombinant lentivirus with high titer and efficient infection of 293T,BCBL-1 and EA.hy926 cells,could be obtained quickly and simply by using the lentivirus vectors system,and Nef expression could be detected in 293T,BCBL-1 and EA.hy926 cells infected by lentivirus-Nef.Furthermore,our study suggested that Nef protein could inhibit the proliferation of BCBL-1 and EA.hy926 cells.更多还原