EGS抑制HCMV UL49基因表达及病毒复制的研究

发布时间：2018-05-15 04:09

本文选题：EGS + 抗病毒　；参考：《暨南大学》2006年博士论文

【摘要】：采用RT-PCR技术，在HCMV(AD169)感染的人胚肺成纤维细胞中检测到了HCMVUL49mRNA，因而首次在RNA水平证实HCMV UL49基因的表达。此外，进一步检测不同感染时间(1h、5h、9h、12h、24h、48h和72h)受染细胞中UL49mRNA的表达情况，初步判断UL49并非即刻早期表达基因(IE)，而可能是一个早期(β2)表达基因。根据HCMV UL49mRNA的序列，借助计算机软件模拟，设计了一组针对HCMV UL49mRNA不同靶位的EGS(共8个，分别命名为EGS1～EGS8)，以此作为筛选库。通过胞外初筛、胞内复筛和胞内抗病毒活性的检测，拟从中筛选出具有抗病毒活性的EGS。1)胞外初筛：首先从HeLa细胞中提取具体外切割活性的RNase P，并以此建立了一种EGS胞外筛选系统。通过该系统证实，所设计的八个EGS中，有六个EGS具有靶向引导切割活性，它们分别是EGS1、EGS3、EGS4、EGS5、EGS6和EGS7，其余的EGS2和EGS8则无明显活性。在具活性的六个EGS中，各EGS的靶向引导切割效率有所不同。其中EGS1、EGS3、EG6和EGS7四个EGS的活性较强，靶向引导切割的效率均超过50％；而EGS4和EGS5的靶向引导切割效率则均低于50％。2)胞内复筛中：首先将UL49基因克隆至pGEFP-N1质粒绿色荧光蛋白基因的上游，以构建UL49-绿色荧光蛋白融合表达载体(pUL49／EGFP-N1)。将EGS与该融合蛋白表达载体以脂质体共转染HeLa细胞(同时以红色荧光质粒pDsRed2-C1，作为转染的内对照)，建立了一种有效的EGS胞内复筛系统。通过荧光显微观察、Typhoon扫描及流式细胞仪检测，证实胞外初筛中活性较强的四个EGS中，只有EGS1、EGS3和EGS6具有明显的胞内靶向抑制活性，而EGS7的活性则较弱。3)胞内抗病毒活性的检测：将初筛及复筛证实有效的EGS分别以脂质体转染HELF细胞，然后再以AD169病毒株感染。在感染24h、48h和72h后，抽提感染细胞的总RNA，以相对荧光定量PCR(β-actin作内参)检测其中HCMVUL49mRNA的含量。结果表明：EGS1、EGS3、EGS6和EGS7对HCMV UL49mRNA表达的抑制率分别为74.87±2.85％、17.85±4.16％、89.77±0.91％和35.40±1.92％。其中，EGS1和EGS6的靶向抑制活性明显高于其余两个EGS(P＜0.01)。进一步以EGS1和EGS6作为研究对象，通过绝对荧光定量PCR的方法检测EGS转染后病毒培养液中HCMV基因组DNA的变化，，以此评价EGS1及EGS6对胞内HCMV复制的抑制活性。结果显示，E6S1和EGS6转染后，病毒的增殖曲线均明显低于未转染EGS的对照组。从而证实了EGS1和EGS6在胞内的抗病毒效应。其中，EGS6的抑制作用较强(抑制率为89.62±2.55％～95.9±1.98％)，EGS1的抑制作用则相对较弱(抑制率为40.25±1.05％～70.85±2.03％)。总之，通过本研究一系列的筛选步骤，获得两种具有胞内抗HCMV活性的EGS序列(EGS1和EGS6)，从而为新型抗HCMV反义药物的进一步研究与开发奠定了基础。
[Abstract]:HCMVUL49mRNAs were detected in human embryonic lung fibroblasts infected with RT-PCR technique, and the expression of HCMV UL49 gene was confirmed for the first time at RNA level. In addition, the expression of UL49mRNA in the infected cells was further detected at different infection time (1h, 5h, 9h, 12h, 24h, 48h, 72h). It was preliminarily determined that UL49 was not an immediate early expressed gene, but an early (尾 _ 2) expression gene. According to the sequence of HCMV UL49mRNA and computer software simulation, a set of EGSs (eight EGS1s EGS8) for different targets of HCMV UL49mRNA were designed and used as the screening library. The extracellular screening, the intracellular screening and the detection of intracellular antiviral activity were carried out. The extracellular screening of EGS.1 with antiviral activity was proposed. Firstly, specific extracellular RNase Ps were extracted from HeLa cells, and a EGS extracellular screening system was established. It was confirmed by the system that six of the eight EGS designs had targeted guided cleavage activity, which were EGS1, EGS3, EGS4, EGS5, EGS6 and EGS7, while the rest EGS2 and EGS8 had no obvious activity. In the six active EGS, the efficiency of targeted guide cutting of each EGS is different. Among them, EGS1, EGS3, EG6 and EGS7 have strong activity, and the efficiency of targeting guided cleavage is more than 50%, while that of EGS4 and EGS5 is lower than that of 50%. 2) the UL49 gene is first cloned into the upstream of the pGEFP-N1 plasmid green fluorescent protein gene. To construct the fusion expression vector of UL49-green fluorescent protein, pUL49 / EGFP-N1. The expression vector of EGS and the fusion protein was co-transfected into HeLa cells with liposome (and the red fluorescent plasmid pDsRed2-C1 was used as the transfection internal control) to establish an effective EGS intracellular screening system. By fluorescence microscopy, Typhoon scanning and flow cytometry analysis showed that of the four EGS with strong activity in the extracellular screen, only EGS1, EGS3 and EGS6 had obvious intracellular targeted inhibitory activities. But the activity of EGS7 was weaker. 3) the detection of intracellular antiviral activity: the EGS which was proved to be effective by primary screening and rescreening was transfected into HELF cells by liposome, and then infected by AD169 virus strain. The total RNAs of infected cells were extracted for 48 h and 72 h after infection, and the content of HCMVUL49mRNA was detected by relative fluorescence quantitative PCR (尾 -actin as internal control). The results showed that the inhibition rates of EGS6 and EGS7 on HCMV UL49mRNA expression were 74.87 卤2.85 and 17.85 卤4.16, respectively, 89.77 卤0.91% and 35.40 卤1.92%, respectively. The targeted inhibitory activities of EGS1 and EGS6 were significantly higher than those of the other two EGS(P < 0.01. Furthermore, EGS1 and EGS6 were used to detect the changes of HCMV genomic DNA in the culture medium after EGS transfection with absolute fluorescence quantitative PCR (PCR) to evaluate the inhibitory activity of EGS1 and EGS6 on the intracellular HCMV replication. The results showed that after transfection of E6S1 and EGS6, the proliferation curve of the virus was significantly lower than that of the control group without EGS transfection. Therefore, the antiviral effects of EGS1 and EGS6 in cells were confirmed. The inhibitory rate of EGS6 was stronger (the inhibitory rate was 89.62 卤2.55) and the inhibitory effect of EGS1 was relatively weak (the inhibitory rate was 40.25 卤1.05 卤70.85 卤2.03). In conclusion, through a series of screening steps in this study, two EGS sequences, EGS1 and EGS6, with intracellular HCMV activity were obtained, which laid a foundation for the further research and development of new antisense drugs against HCMV.
【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346

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