人巨细胞病毒UL49基因在病毒复制过程中的功能初步研究

发布时间：2018-11-17 19:27

【摘要】： 目的：UL49基因是人巨细胞病毒必需基因,编码病毒颗粒皮层蛋白,本课题对UL49基因在病毒复制过程中的功能进行了初步研究。方法：(1)以HCMV病毒感染人包皮成纤维细胞(HFF),在感染后不同时间点收获细胞,以即刻早期IE基因、早期E基因、晚期L基因表达时相作为对照,利用RT-PCR和Western Blot方法确定UL49基因mRNA和蛋白表达时相。(2)化学合成以UL49基因mRNA为靶的EGS和阴性对照EGS,转染HFF细胞、感染HCMV病毒,利用荧光定量PCR和Western Blot检测EGS对UL49基因的表达抑制,进而检测细胞中gB蛋白、IE2-86蛋白、pp28蛋白的表达水平变化。(3)根据上述实验结果,比较UL49基因沉默后,病毒DNA总量在感染后24h、48h、72h变化,探讨UL49基因在病毒DNA复制中的功能。结果：(1)UL49基因是早期E-β2基因,编码蛋白在病毒感染24h能够检测到。(2)pUL49蛋白的表达下调,不影响IE2-86蛋白、gB蛋白的表达,但是使pp28蛋白表达下调。(3)UL49基因沉默不影响细胞内病毒总DNA含量,UL49基因与病毒DNA复制功能无关。结论：UL49基因不参与病毒DNA复制。pUL49蛋白发挥作用的时期为病毒DNA复制后L-γ2基因的表达或者病毒的组装过程。
[Abstract]:Objective: UL49 gene is an essential gene of human cytomegalovirus and encodes virus granular cortical protein. The function of UL49 gene in viral replication was studied in this paper. Methods: (1) Human prepuce fibroblasts (HFF),) infected with HCMV virus were harvested at different time points after infection. The expression of immediate early IE gene, early E gene and late L gene were used as controls. RT-PCR and Western Blot methods were used to determine the expression phase of mRNA and protein of UL49 gene. (2) EGS targeting UL49 gene mRNA and EGS, as negative control were chemically synthesized and transfected into HFF cells and infected with HCMV virus. Fluorescence quantitative PCR and Western Blot were used to detect the inhibition of UL49 gene expression by EGS, and then the expression levels of gB protein, IE2-86 protein and pp28 protein were detected. (3) according to the above results, UL49 gene silencing was compared. The total amount of virus DNA changed at 24 h, 48 h and 72 h after infection. The function of UL49 gene in viral DNA replication was studied. Results: (1) UL49 gene is an early E- 尾 2 gene, and the encoded protein can be detected at 24 h after infection. (2) the expression of pUL49 protein is down-regulated, and the expression of IE2-86 protein and gB protein is not affected. However, the expression of pp28 protein was down-regulated. (3) UL49 gene silencing did not affect the total DNA content of the virus, and the UL49 gene had nothing to do with the replication function of the virus DNA. Conclusion: UL49 gene does not participate in the replication of virus DNA. The stage of pUL49 protein acting is the expression of L- 纬 2 gene or the assembly of virus after DNA replication.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373

【共引文献】