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目的:利用慢病毒表达载体将HIV-1Vif基因导入原发性渗出性淋巴瘤(PEL)细胞BCBL-1中使之持续表达目的蛋白Vif,并检测Vif对其中潜伏KSHV裂解性周期复制的影响。方法:自表达质粒pCI-neo-Vif中扩增出Vif基因,插入到pHAGE-CMV-MCS-IzsGreen中构建成慢病毒载体pHAGE-Vif,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。荧光显微镜观察293T细胞中绿色荧光蛋白(GFP)表达情况;收集培养上清经0.45μm滤器过滤后即获得病毒悬液。梯度稀释法测定病毒滴度,感染293T细胞,48h或72h后进行RT-PCR和Western blot检测Vif基因的转录和表达情况。以MOI为1.0的病毒量感染靶细胞BCBL-1,利用Westernblot技术检测Vif蛋白的表达,同时检测KSHVvIL-6和Rta的蛋白表达水平,初步探讨Vif对KSHV复制的影响。结果:限制性内切酶检测和基因测序证实成功构建了携带Vif基因的慢病毒表达载体,滴度为4×107efu/ml。以MOI为1.0的重组慢病毒感染靶细胞BCBL-172h后,能够检测到外源基因Vif的蛋白表达。Western blot结果初步显示,Vif能够下调vIL-6和Rta的蛋白表达水平。结论:成功构建了含Vif基因的慢病毒表达载体,获得的病毒能够有效感染BCBL-1细胞,并在其中大量表达目的蛋白。初步结果提示,Vif能够下调vIL-6和Rta的蛋白表达水平,对KSHV裂解性周期复制可能起到抑制作用。
OBJECTIVE: To introduce the HIV-1 Vif gene into BCBL-1 of primary exudative lymphoma (PEL) cells by lentiviral expression vector to express the target protein Vif continuously and to detect the effect of Vif on the periodical replication of latent KSHV . Methods: The Vif gene was amplified from the expression plasmid pCI-neo-Vif and inserted into pHAGE-CMV-MCS-IzsGreen to construct the lentiviral vector pHAGE-Vif. The liposome was used to ligate it with the packaging plasmid psPAX2 and envelope plasmid 293T cells were co-transfected with pMD2.G. Fluorescence microscopy was used to observe the expression of green fluorescent protein (GFP) in 293T cells. The supernatant was collected and filtered through a 0.45 μm filter to obtain a virus suspension. The virus titer was determined by gradient dilution method and 293T cells were infected. The transcription and expression of Vif gene were detected by RT-PCR and Western blot at 48h or 72h. The target cell line BCBL-1 was infected with a MOI of 1.0, and the protein expression of Vif was detected by Western blotting. The protein expression levels of KSHVvIL-6 and Rta were also detected, and the effect of Vif on KSHV replication was also investigated. Results: The restriction endonuclease assay and gene sequencing confirmed the successful construction of the lentiviral vector carrying Vif gene with a titer of 4 × 107efu / ml. The target gene BCBL-172h was infected with recombinant lentivirus at the MOI of 1.0, and the protein expression of the exogenous gene Vif was detected. Western blot results initially showed that Vif down-regulated the protein expression of vIL-6 and Rta. CONCLUSION: The lentiviral vector containing Vif gene has been successfully constructed. The obtained virus can effectively infect BCBL-1 cells and express a large amount of the target protein therein. Preliminary results suggest that Vif can down-regulate the protein expression of vIL-6 and Rta, which may inhibit KSHV cell cycle replication.