论文部分内容阅读
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PmeⅠ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PacⅠ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3.0×108efu/ml。以MOI为5的重组腺病毒感染24 h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 a recombinant adenovirus containing HIV-1 viral protein R (Vpr) gene by AdEasy adenovirus vector system so that it can efficiently infect target cell prostate cancer cell line PC-3 and express Vpr in it. Methods: The Vpr gene was amplified from the expression vector pCI-neo-Vpr and inserted into pAdTrack-CMV to construct the adenovirus shuttle plasmid pAdTrack-Vpr. After digestion with restriction endonuclease PmeⅠ, The guide method and its adenovirus backbone plasmid pAdEasy co-transformed into BJ5183 Escherichia coli. The homologous recombination plasmids were selected and digested with Pac Ⅰ to recover the large fragment, which was then transfected into packaging cells AD293. The expression of green fluorescent protein (GFP) in AD293 cells was observed by fluorescence microscopy. The first generation of recombinant virus supernatant was collected and infected with AD293 cells to obtain the second generation of virus, so repeated infection of AD293 cells 3 to 4 rounds, so that a large number of virus amplification. After the virus titer was determined by gradient dilution method, the target cells PC-3 were infected by the virus with multiplicity of infection (MOI) of 1, 5 and 10 respectively. The expression of GFP was observed by fluorescence microscopy. The expression of GFP was detected by RT-PCR and Western blot respectively Detection of transcription and expression of the target gene from the mRNA and protein levels. Results: The recombinant adenovirus carrying Vpr gene was confirmed by restriction endonuclease assay, GFP expression and virus supernatant PCR with a titer of 3.0 × 108efu / ml. After being infected with recombinant adenovirus with an MOI of 5 for 24 h, more than 80% of the target cells could express GFP. The transcription and expression of the target gene Vpr can be detected simultaneously by RT-PCR and Western blotting. CONCLUSION: The recombinant adenovirus containing Vpr gene is successfully constructed and the virus can effectively infect PC-3 cells so that the gene of interest is abundantly expressed therein. To further investigate the effect of Vpr protein on PC-3 tumor cells and its possible signaling pathways Foundation.