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...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。展开更多
Viral protein R(Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1(HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cyc...Viral protein R(Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1(HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cycle arrest, activating the NF-κB pathway, and promoting viral reverse transcription, might be interrelated. To test this hypothesis, a panel of Vpr mutants were investigated for their ability to induce G2/M arrest and to activate the NF-κB pathway. The results showed that the Vpr mutants that failed to activate NF-κB also lost the activity to induce G2/M arrest, which suggests that inducing G2/M arrest via Vpr depends at least partially on the activation of NF-κB. This latter possibility is supported by data showing that knocking down the key factors in the NF-κB pathway – p65, Rel B, IKKα, or IKKβ– partially rescued the G2/M arrest induced by Vpr.Our results suggest that the NF-κB pathway is probably involved in Vpr-induced G2/M cell cycle arrest.展开更多
文摘目的：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。
基金supported by grants from the Chinese Ministry of Health (2012ZX10001006)the National Natural Science Foundation of China (81271812 and 31370182)+1 种基金111 Project (B08011)the Postgraduate Scholarship Program of the China Scholarship Council
文摘Viral protein R(Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1(HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cycle arrest, activating the NF-κB pathway, and promoting viral reverse transcription, might be interrelated. To test this hypothesis, a panel of Vpr mutants were investigated for their ability to induce G2/M arrest and to activate the NF-κB pathway. The results showed that the Vpr mutants that failed to activate NF-κB also lost the activity to induce G2/M arrest, which suggests that inducing G2/M arrest via Vpr depends at least partially on the activation of NF-κB. This latter possibility is supported by data showing that knocking down the key factors in the NF-κB pathway – p65, Rel B, IKKα, or IKKβ– partially rescued the G2/M arrest induced by Vpr.Our results suggest that the NF-κB pathway is probably involved in Vpr-induced G2/M cell cycle arrest.
