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目的:研究人类免疫缺陷病毒-1(HIV-1)Rev蛋白的表达水平对原发性渗出性淋巴瘤(PEL)细胞BCBL-1中卡波济肉瘤相关疱疹病毒(KSHV)裂解性周期复制与潜伏的影响。方法:自表达质粒pCI-neo-Rev中扩增出Rev基因,插入到pHAGE-CMV-MCS-IZsGreen中构建成慢病毒载体pLenti-Rev,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(IZsGreen)表达;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并感染细胞,RT-PCR和Western blot检测Rev在293T细胞中的表达。用慢病毒感染BCBL-1细胞,同时将表达质粒pCI-neo-Rev转染该细胞,采用Western blot分别检测Rev以及KSHV Rta和vIL-6蛋白的表达;Real-timePCR进一步从mRNA水平上定量验证Rev对KSHV复制的影响。结果:限制性内切酶酶切反应和基因测序证实成功构建了携带Rev基因的慢病毒表达载体,病毒滴度为2×107TU/ml。重组慢病毒感染BCBL-1细胞72h后,能够检测到Rev蛋白的表达。Western blot结果显示,低水平表达的Rev能够上调KSHV裂解期蛋白Rta、vIL-6的表达,而高水平表达的Rev则能够抑制Rta和vIL-6的表达。Real-time PCR进一步验证了该结果,表明高水平表达的Rev能够显著抑制BCBL-1中Rta的mRNA转录,96h尤为明显。结论:成功构建了含Rev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Rev蛋白。Rev蛋白的表达水平对KSHV的复制与潜伏可能起到复杂的调控作用。
OBJECTIVE: To study the effect of the Rev-protein level of human immunodeficiency virus-1 (HIV-1) on the replication of the Kaposi’s sarcoma-associated herpesvirus (KSHV) in BCL-1 cells of primary exudative lymphoma (PEL) And the potential impact. METHODS: The Rev gene was amplified from the expression plasmid pCI-neo-Rev and inserted into pHAGE-CMV-MCS-IZsGreen to construct lentiviral vector pLenti-Rev. The recombinant plasmid was ligated with plasmid psPAX2 and envelope plasmid 293T cells were co-transfected with pMD2.G. Fluorescence microscopy observed green fluorescent protein (IZsGreen) expression in 293T cells; collected supernatant was filtered through a 0.45μm filter to obtain a virus suspension. The virus titer was determined by gradient dilution method and the cells were infected. The expression of Rev in 293T cells was detected by RT-PCR and Western blot. BCBL-1 cells were infected with lentivirus, and the expression plasmid pCI-neo-Rev was transfected into the cells. The expression of Rev and KSHV Rta and vIL-6 protein were detected by Western blot. Real-time PCR was further used to quantitatively verify the mRNA level Effect of Rev on KSHV replication. Results: Restriction endonuclease digestion and gene sequencing confirmed the successful construction of lentiviral vector carrying Rev gene. The virus titer was 2 × 107TU / ml. The expression of Rev protein was detected 72 h after infection with recombinant lentivirus in BCBL-1 cells. Western blot results showed that Rev at low level increased the expression of Rta and vIL-6 at the KSHV cleavage stage, whereas Rev at high level inhibited Rta and vIL-6 expression. Real-time PCR further validated the results, indicating that high level of expression of Rev can significantly inhibit mRNA transcription of Rta in BCBL-1, especially at 96h. CONCLUSION: The lentiviral vector containing Rev gene was successfully constructed. The obtained virus can effectively infect BCBL-1 cells and express abundant Rev protein. The expression level of Rev protein may play a complex regulatory role in the replication and lodging of KSHV.