摘要
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.
出处
《江苏大学学报(医学版)》
CAS
2010年第4期286-291,共6页
Journal of Jiangsu University:Medicine Edition
基金
教育部新世纪优秀人才支持计划资助项目(NCET-05-0506)