靶向ICP4的RNA干扰对HSV-1病毒复制能力影响

发布时间：2018-07-10 10:11

本文选题：单纯疱疹病毒1型 + 即早蛋白4　；参考：《郑州大学》2010年硕士论文

【摘要】： 单纯疱疹病毒1型(HSV-1)为双链包膜DNA病毒,具有亲神经的特性,在人群中感染广泛。HSV-1感染外周神经后可逆行跨越神经元进入中枢神经系统并建立潜伏感染,病毒裂解后引起中枢神经系统严重损伤。HSV-1基因组表达有很高时序性,分即早基因(IE),早期基因(E)和晚期基因(L)。作为IE基因产物之一的即早蛋白ICP4可刺激E基因表达和DNA合成,诱导L基因的表达,是HSV-1基因表达从而引发感染的关键激活因子。因此,有效抑制ICP4的表达可以通过阻止HSV-1的复制达到治疗HSV-1感染性疾病的目的。RNA干扰(RNAi)是一种细胞本身固有的对抗外源基因侵犯的自我保护现象,双链RNA(dsRNA)分子在mRNA水平关闭相应序列及基因的表达使其沉默。在此基础上建立的RNAi技术不仅仅是一种经济、快捷、高效的抑制基因表达的技术手段,更为研究者在基因功能测定和基因治疗等方面开辟了新思路。本实验构建表达ICP4 siRNA (小干扰RNA)的重组真核慢病毒质粒PLKO.1-puro-ICP4-siRNA,将产生的重组病毒颗粒感染Vero细胞,建立稳定表达ICP4 siRNA的重组Vero-ICP4-siRNA细胞系；进一步探讨干扰HSV-1 ICP4基因后对HSV-1复制能力的影响,为临床上运用RNAi治疗HSV-1感染性疾病提供思路和实验依据。目的： 1建立稳定表达ICP4特异性siRNA的重组细胞系。 2初步分析靶向ICP4的siRNA对HSV-1在Vero细胞中复制能力的影响。方法： 1基因工程技术构建重组慢病毒质粒PLKO.1-puro-ICP4-siRNA,双酶切及测序鉴定。 2三质粒共转染293T细胞产生重组慢病毒颗粒。 3重组病毒颗粒感染Vero细胞,嘌呤霉素筛选法建立ICP4-siRNA表达阳性的重组细胞系。 4采用Realtime PCR和Western blot鉴定重组Vero-ICP4-siRNA细胞系。 5通过观察HSV感染重组Vero细胞后CPE(细胞病变效应)表达和TCID50(半数组织培养感染剂量)法测定HSV-1滴度,检测靶向ICP4的siRNAs对HSV-1病毒复制能力的影响。结果： 1成功构建重组真核慢病毒质粒PLKO.1-puro-ICP4-siRNA。 2获得有效浓度的稳定表达ICP4的siRNAs的重组慢病毒颗粒。 3成功建立稳定表达ICP4特异性siRNA的重组细胞系。 4在mRNA和蛋白水平证实重组Vero-ICP4-siRNA细胞中的HSV-1 ICP4表达被抑制。 5感染野生型HSV-1后,siRNA组Vero细胞的CPE表达率与阴性对照组比较有明显降低。进一步采用TCID50法测定病毒滴度并对TCID值进行AVONA统计学分析发现,siRNA组HSV滴度与对照组存在显著差异(P0.001), siRNA组低于对照组；且针对两个靶点的siRNA联合组HSV-1病毒滴度低于任何单一抑制组(P0.001)。结论： 1成功建立稳定表达抗ICP4 siRNA的重组Vero细胞系。 2 SiRNA能够有效地干扰HSV-1 ICP4基因表达,且不同位点联合干扰对HSV-1 ICP4基因的表达有协同的抑制效果,进而对HSV-1病毒复制有协同抑制效果。
[Abstract]:Herpes simplex virus type 1 (HSV-1) is a double-strand envelope DNA virus, which has the characteristics of neurobiosis. HSV-1 can retrograde into the central nervous system (CNS) and establish latent infection after infection of peripheral nerves with HSV-1. The genome expression of HSV-1 was highly sequential, I. E. early gene (IE), early gene (E) and late gene (L). ICP4, one of the products of IE gene, can stimulate the expression of E gene and DNA synthesis, induce the expression of L gene, and is the key activator of HSV-1 gene expression. Therefore, the effective inhibition of ICP4 expression can be used to treat HSV-1 infectious diseases by preventing HSV-1 replication. RNA interference (RNAi) is an inherent self-protection phenomenon of cells against foreign gene invasion. Double stranded RNA (dsRNA) silences the sequence and gene expression at the mRNA level. The RNAi technology established on this basis is not only an economical, fast and efficient technique to inhibit gene expression, but also opens up new ideas for gene function determination and gene therapy. In this experiment, the recombinant eukaryotic lentivirus plasmid PLKO.1-puro-ICP4-siRNAs expressing ICP4 siRNA was constructed. The recombinant virus particles were infected into Vero cells, and a recombinant Vero-ICP4-siRNA cell line was established which expressed ICP4 siRNA stably. To further investigate the effect of interference with HSV-1 ICP4 gene on HSV-1 replication ability, and to provide an experimental basis for the clinical treatment of HSV-1 infectious diseases with RNAi. Aim: 1 to establish a recombinant cell line stably expressing ICP4 specific siRNA. 2 to analyze the effect of ICP4 targeting siRNA on HSV-1 replication in Vero cells. Methods: 1Recombinant lentivirus plasmids PLKO.1-puro-ICP4-siRNAs were constructed by genetic engineering technique and identified by double enzyme digestion and sequencing. 2Recombinant lentivirus particles were produced by co-transfection of three plasmids into 293T cells. A recombinant cell line with positive expression of ICP4-siRNA was established by purine mycin screening. 4 Recombinant Vero-ICP4-siRNA cell line was identified by Realtime PCR and Western blot. 5 CPE (fine) after HSV infection of recombinant Vero cells was observed. The expression of cytopathic effect and the titer of HSV-1 were measured by TCID50 method. The effect of siRNAs targeting ICP4 on the replication ability of HSV-1 virus was detected. Results: 1Recombinant lentivirus particles were successfully constructed into recombinant eukaryotic lentivirus plasmid PLKO.1-puro-ICP4-siRNA.2 with stable concentration of siRNAs expressing ICP4. 3 To establish a recombinant cell line stably expressing ICP4-specific siRNA. 4 the expression of HSV-1 ICP4 in recombinant Vero-ICP4-siRNA cells was confirmed at mRNA and protein levels. 5 Vero of siRNA group after infection with wild-type HSV-1 was confirmed by inhibition of HSV-1 ICP4 expression in recombinant Vero-ICP4-siRNA cells. The expression rate of CPE in the cells was significantly lower than that in the negative control group. The titer of HSV in siRNA group was significantly higher than that in control group (P0.001), and the titer of HSV in siRNA group was lower than that in control group (P0.001). The titer of HSV-1 virus in siRNA combination group was lower than that in any single inhibition group (P0.001). Conclusion: 1Recombinant Vero cell line with stable expression of anti-ICP4 siRNA was successfully established. 2SiRNA could effectively interfere with the expression of HSV-1 ICP4 gene. The co-interference of different sites had synergistic inhibitory effect on HSV-1 ICP4 gene expression and then on HSV-1 virus replication.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

【共引文献】