重组腺病毒HIV-1 vpr基因的构建、表达及其生物学功能的初步研究

发布时间：2018-03-11 22:10

本文选题：HIV-1　切入点：vpr　出处：《暨南大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：研究HIV-1 vpr基因编码的病毒蛋白R(Viral protein R，Vpr)对CD4~+T淋巴细胞系C8166生物学行为的影响，为进一步了解Vpr的功能和作用机制，及今后研究Vpr引起整个CD4~+T淋巴细胞蛋白组的漂移奠定基础。方法：通过同源重组构建重组腺病毒质粒，用脂质体法将重组质粒转染至包装细胞HEK293，获得重组腺病毒Ad-vpr，荧光显微镜观察Ad-vpr感染C8166细胞，Westernblotting鉴定Vpr在Ad-vpr感染的C8166细胞内的特异性表达，流式细胞术等检测Vpr对C8166细胞的细胞膜完整性，，细胞周期，凋亡等生物学行为的影响。透射电镜、激光共聚焦显微镜观察Vpr诱导的C8166细胞形态的改变。结果：构建了重组腺病毒Ad-vpr，证实了Vpr在Ad-vpr感染的C8166细胞内的表达，Annexin V-APC染色流式细胞术分析结果表明；Ad-vpr感染48h后C8166细胞GFP~+细胞早期凋亡率与重组空载体感染组(Ad-vector组)相比无明显差别；而Ad-vpr感染72h后，C8166 GFP~+细胞早期凋亡率显著高于Ad-vector组。PI染色流式细胞术检测膜完整性表明：Ad-vpr感染48h的C8166细胞与Ad-vector感染48h的C8166细胞相比，细胞膜完整性明显不能维持。PI染色流式细胞术检测细胞周期结果显示，培养48h的C8166细胞，Ad-vpr感染组晚期凋亡率及S期细胞百分数显著高于Ad-vector组，而
[Abstract]:Objective:. To study the effect of HIV-1 vpr gene encoded virus protein rvpr) on the biological behavior of CD4T lymphocyte line C8166, and to lay a foundation for further understanding the function and mechanism of Vpr and for studying the drift of whole CD4T lymphocyte protein group induced by Vpr in the future. Methods:. The recombinant adenovirus plasmid was constructed by homologous recombination and transfected into the packaging cell line HEK293 by liposome method. The recombinant adenovirus Ad-vpra was obtained. The specific expression of Vpr in Ad-vpr infected C8166 cells was identified by Western blotting with fluorescence microscope. The effects of Vpr on cell membrane integrity, cell cycle and apoptosis of C8166 cells were detected by flow cytometry. The morphological changes of C8166 cells induced by Vpr were observed by transmission electron microscope and confocal laser microscope. Results:. The recombinant adenovirus Ad-vpr1 was constructed, and the expression of Vpr in Ad-vpr infected C8166 cells was confirmed by flow cytometry. The results of flow cytometry showed that the early apoptosis rate of C8166 cells infected by Ad-vpr was not significantly different from that of Ad-vector infected C8166 cells. However, the early apoptosis rate of C8166 GFP~ ~ cells infected with Ad-vpr was significantly higher than that of C8166 cells infected with Ad-vector for 48 h after infection with Ad-vector. The membrane integrity of C8166 cells infected with Ad-vpr was significantly higher than that of C8166 cells infected with Ad-vector for 48 h. The results of cell cycle analysis by flow cytometry showed that the late apoptosis rate and S phase percentage of C8166 cells infected with Ad-VPR for 48 h were significantly higher than those in Ad-vector group.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

【相似文献】