HIV-1型病毒蛋白R基因表达载体的构建及其在前列腺癌细胞系PC-3中的表达

Construction of the recombinant expression adenovirus vector carrying HIV-1 viral protein R gene and its expression in PC-3 cell line

目的：利用AdEasy腺病毒载体系统构建含HIV-1病毒蛋白R（viral protein R，Vpr）基因的重组腺病毒，使之有效感染靶细胞前列腺癌细胞系PC-3，并在其中表达Vpr。方法：自表达载体pCI—neo—Vpr中扩增出Vpr基因，插入到pAdTrack—CMV中构建成腺病毒穿梭质粒pAdTrack—Vpr，经限制性内切酶Pme I酶切线性化后，利用磷酸钙介导法将其与腺病毒骨架质粒pAdEasy共同转化到BJ5183大肠埃希菌。挑选同源重组质粒，经Pac I酶切后回收大片段，并将其转染包装细胞AD293。利用荧光显微镜观察AD293细胞中绿色荧光蛋白（GFP）表达。收集第一代重组病毒上清，使之感染AD293细胞得到第二代病毒，如此反复感染AD293细胞3—4轮，使病毒大量扩增。梯度稀释法测定病毒滴度后，分别以感染复数（MOI）为1、5、10的病毒量感染靶细胞PC-3，荧光显微镜观察细胞中GFP表达，并利用RT＋PCR和蛋白质印迹技术分别从mRNA和蛋白水平检测目的基因的转录与表达情况。结果：经限制性内切酶检测、GFP表达和病毒上清液PCR证实成功构建了携带Vpr基因的重组腺病毒，滴度为3．0×10^8efu／ml。以MOI为5的重组腺病毒感染24h后，80％以上的靶细胞能够表达GFP，RT—PCR和蛋白质印迹能够同时检测到目的基因Vpr的转录与表达。结论：成功构建含Vpr基因重组腺病毒，并且病毒能够有效感染PC-3细胞，使得目的基因在其中获得大量表达，为进一步研究Vpr蛋白对PC-3肿瘤细胞的影响及其可能涉及的信号通路奠定了基础。

Objective： To construct the recombinant adenovirus containing HIV-1 viral protein R （Vpr） gene, and investigate the expression of Vpr protein in PC-3 cells. Methods： The fragment of Vpr gene from expression vector pCI-neo-Vpr was cloned into the shuttle plasmid pAdTrack-CMV. With the backbone plasmid pAdEasy-1 and the Pine [ linearized shuttle plasmid pAdTraek-Vpr, the recombinant adenoviral plasmid was generated from the homologous recombination in the E. coli BJ5183. The adenoviruses were packaged in the AD293 cells by transecting the Pac I linearized recombinant adenoviral plasmid, and were amplified by infecting AD293 cells, repetitively for 3 - 4 rounds. Then the viral titer was evaluated by green fluorescent protein （GFP） under fluorescence microscope. After infecting the PC-3 cells with the recombinant adenovirus at MOI of 1,5 and 10, the mRNA and protein expression of Vpr in PC-3 ceils was detected by RT-PCR and Western blot, respectively. Results： Confirmed by the restriction enzyme digestion analysis, the recombinant adenovirus vector carrying Vpr was constructed successfully with the viral titer 3.0 × 10^8efu/ml. And more than 80% PC-3 cells could be infected by the adenovirus at the MOI of 5. In addition, the expression of Vpr in PC-3 cells infected with AdEasy-GFP-Vpr was detectable by RT-PCR and Western blot. Conclusion： Recombinant adenoviruses with high titer and efficient infection of PC-3 cells could be obtained quickly and simply by using AdEasy system, and Vpr expression could be observed in PC-3 cells infected with AdEasy-GFP-Vpr. The results of this study lay the foundation for further studying on the role of Vpr protein in its anti-tumor activity and related signaling pathways.