RNA干扰特异抑制单纯疱疹病毒1型衣壳蛋白基因及US12基因的研究
发布时间：2018-07-29 14:09
【摘要】：目的：筛选高效沉默HSV-1病毒衣壳相关基因的siRNA;研究siRNA沉默目的基因后对HSV-Ⅰ感染与增殖的影响,为抗单纯疱疹病毒药物开发提供依据；探究衣壳蛋白与宿主细胞,衣壳蛋白与信号通路的相互关系；筛选高效沉默HSV-1 US12基因的siRNA,研究siRNA沉默US12基因后对HSV-Ⅰ增殖的影响。 方法：设计并化学合成针对7个HSV-1衣壳蛋白基因的siRNA,转染Vero细胞,通过荧光定量PCR检测相关siRNA对目的基因mRNA表达的抑制效果。空斑减数实验评价siRNA抑制目的基因对HSV-1感染与增殖的影响。形态学观察siRNA抑制感染细胞病变的情况。透射电镜观察siRNA干扰对核内病毒衣壳复制及出核的影响。构建可以用于高效筛选目的基因及蛋白表达的pEGFP-N1-US12融合表达质粒。采用QUICK技术研究VP5相互作用蛋白。 结果：共转染实验筛选出高效抑制UL18、UL19、UL26、UL26.5、UL35、UL38mRNA表达的siRNA,但空斑减数实验发现只有针对UL18,UL19基因的siRNA对HSV-1的增殖有显著抑制效果。siRNA干扰UL19基因能够显著抑制细胞病变情况。透射电镜观察发现干扰UL19基因可以降低核内病毒衣壳数量,抑制病毒衣壳释放。成功构建可以用于高效筛选目的基因及蛋白表达的pEGFP-N1-US12融合表达质粒。通过QUICK技术筛选出5个与VP5相互作用作用蛋白。 结论：设计合成的siRNA可以有效降低HSV-1衣壳蛋白各相关基因的表达,针对UL18和UL19的siRNA能显著地抑制病毒的感染与增殖,因此推测UL18基因与UL19基因是开发HSV-1抗病毒药物的潜在靶点。RNA干扰US12基因能显著降低US12的mRNA水平和融合蛋白表达,但是对HSV-1的体外增殖无显著影响,这表明US12表达的立即早期蛋白ICP47的功能可能与HSV-1体外增殖无直接相关。
[Abstract]:Objective: to screen siRNA for highly efficient silencing of HSV-1 virus capsid related genes, to study the effect of siRNA silent target gene on HSV- I infection and proliferation, to provide a basis for the development of anti herpes simplex virus drugs, to explore the relationship between capsid protein and host cells, capsid protein and signal pathway, and to screen the efficient and silent HSV-1 US12 gene. SiRNA, to study the effect of siRNA silencing US12 gene on the proliferation of HSV- I.
Methods: siRNA was designed and synthesized by chemical synthesis of 7 HSV-1 capsid protein genes, Vero cells were transfected and the inhibitory effect of related siRNA on the expression of mRNA was detected by fluorescence quantitative PCR. The effect of siRNA inhibition gene on HSV-1 infection and proliferation was evaluated by space spot subtraction. The effect of siRNA interference on the replication and nucleation of viral capsid in the nucleus was observed by transmission electron microscopy. The pEGFP-N1-US12 fusion expression plasmid which could be used to efficiently screen the expression of the target gene and protein was constructed. The QUICK technique was used to study the VP5 interaction protein.
Results: the siRNA of UL18, UL19, UL26, UL26.5, UL35, UL38mRNA expressed effectively was screened by CO transfection experiment, but only UL18, siRNA of UL19 gene had significant inhibitory effect on HSV-1 proliferation. In order to reduce the number of viral capsid and inhibit the release of viral capsid, the pEGFP-N1-US12 fusion expression plasmid, which can be used to efficiently screen the target gene and protein expression, was successfully constructed. 5 proteins interacting with VP5 were screened by QUICK technology.
Conclusion: the synthesized siRNA can effectively reduce the expression of HSV-1 capsid protein related genes, and the siRNA of UL18 and UL19 can significantly inhibit virus infection and proliferation. Therefore, it is speculated that the UL18 gene and UL19 gene are potential targets for developing HSV-1 antiviral drugs,.RNA interference US12 gene can significantly reduce US12 mRNA level and fusion. The protein expression has no significant effect on the proliferation of HSV-1 in vitro, which indicates that the function of the immediate early protein ICP47 expressed by US12 may not be directly related to the proliferation of HSV-1 in vitro.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373
本文编号：2152924
[Abstract]:Objective: to screen siRNA for highly efficient silencing of HSV-1 virus capsid related genes, to study the effect of siRNA silent target gene on HSV- I infection and proliferation, to provide a basis for the development of anti herpes simplex virus drugs, to explore the relationship between capsid protein and host cells, capsid protein and signal pathway, and to screen the efficient and silent HSV-1 US12 gene. SiRNA, to study the effect of siRNA silencing US12 gene on the proliferation of HSV- I.
Methods: siRNA was designed and synthesized by chemical synthesis of 7 HSV-1 capsid protein genes, Vero cells were transfected and the inhibitory effect of related siRNA on the expression of mRNA was detected by fluorescence quantitative PCR. The effect of siRNA inhibition gene on HSV-1 infection and proliferation was evaluated by space spot subtraction. The effect of siRNA interference on the replication and nucleation of viral capsid in the nucleus was observed by transmission electron microscopy. The pEGFP-N1-US12 fusion expression plasmid which could be used to efficiently screen the expression of the target gene and protein was constructed. The QUICK technique was used to study the VP5 interaction protein.
Results: the siRNA of UL18, UL19, UL26, UL26.5, UL35, UL38mRNA expressed effectively was screened by CO transfection experiment, but only UL18, siRNA of UL19 gene had significant inhibitory effect on HSV-1 proliferation. In order to reduce the number of viral capsid and inhibit the release of viral capsid, the pEGFP-N1-US12 fusion expression plasmid, which can be used to efficiently screen the target gene and protein expression, was successfully constructed. 5 proteins interacting with VP5 were screened by QUICK technology.
Conclusion: the synthesized siRNA can effectively reduce the expression of HSV-1 capsid protein related genes, and the siRNA of UL18 and UL19 can significantly inhibit virus infection and proliferation. Therefore, it is speculated that the UL18 gene and UL19 gene are potential targets for developing HSV-1 antiviral drugs,.RNA interference US12 gene can significantly reduce US12 mRNA level and fusion. The protein expression has no significant effect on the proliferation of HSV-1 in vitro, which indicates that the function of the immediate early protein ICP47 expressed by US12 may not be directly related to the proliferation of HSV-1 in vitro.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373
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