本文關鍵詞：EV71病毒抑制RIG-Ⅰ泛素化逃避宿主天然免疫的機制研究　出處：《黑龍江八一農(nóng)墾大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： EV71 RIG-Ⅰ IFN-β 泛素化 天然免疫

【摘要】：腸道病毒71型(Enteroviruses 71,EV71)屬于小核糖核酸病毒科,腸道病毒屬,腸道病毒A型的成員,是引起嬰幼兒手足口病的重要病原體。近年來,EV71病毒在全世界多次爆發(fā)與流行,造成數(shù)萬人的死亡。EV71病毒侵入細胞之后,病毒基因組復制形成雙鏈RNA分子,會被宿主細胞的模式識別受體RIG-Ⅰ識別,隨后RIG-Ⅰ受體分子在泛素連接酶作用下被泛素化,從而啟動下游I型干擾素信號通路。本課題組前期研究發(fā)現(xiàn),EV71病毒感染可以抑制宿主RIG-Ⅰ受體的泛素化水平,但是抑制RIG-Ⅰ泛素化是否最終介導了EV71病毒逃避宿主天然免疫尚不清楚。因此,本論文將以這一發(fā)現(xiàn)為切入點,闡明EV71病毒抑制RIG-Ⅰ泛素化逃避宿主天然免疫這一科學問題,為EV71病毒逃避宿主天然免疫理論提出新的觀點。為了研究EV71病毒在RD細胞內的復制情況,EV71病毒感染RD細胞24小時之后,采用光學顯微鏡和熒光顯微鏡在不同時間點觀察細胞病變。結果顯示,EV71病毒感染6小時,檢測到大量病毒蛋白表達,且細胞出現(xiàn)明顯病變。在確定感染時間點的基礎上,細胞轉染IFN-β-Luc質粒之后,采用雙熒光素酶報告基因系統(tǒng),檢測EV71病毒感染對RD細胞IFN-β啟動子活性的影響。結果顯示,EV71(MOI=20)病毒顯著降低RD細胞IFN-β的啟動子活性。為了確認EV71病毒對IFN-β的抑制作用,采用RT-PCR方法檢測病毒對IFN-β轉錄水平的影響。結果顯示,EV71病毒感染顯著抑制細胞IFN-β的轉錄。干擾素調節(jié)基因3(IRF3)的入核對于活化宿主天然免疫是必不可少的,為了證實EV71病毒通過抑制IRF3的入核拮抗I型干擾素通路的可能性,RD細胞轉染pEGFP-IRF3質粒之后,采用免疫熒光技術觀察IRF3的入核情況。結果顯示,EV71病毒感染明顯抑制了IRF3的入核。干擾素刺激基因(ISG)是發(fā)揮抗病毒作用的最終效應分子,為了研究EV71病毒對ISGs的影響,在EV71病毒感染RD細胞之后,采用RT-PCR方法檢測病毒ISG54、ISG56以及IFN-β的轉錄水平,結果顯示,EV71病毒感染顯著下調RD細胞IFN-β、ISGs的mRNA轉錄水平。為了研究EV71病毒對RIG-Ⅰ受體泛素化的影響,EV71病毒感染RD細胞之后,采用Western Blot方法檢測細胞內源性RIG-Ⅰ的泛素化水平。結果顯示,EV71病毒感染顯著抑制細胞內源性RIG-Ⅰ的泛素化,且表現(xiàn)為時間依賴性。為了證實EV71病毒對RIG-Ⅰ的抑制作用,RD細胞共轉Flag-RIG-Ⅰ和HA-Ub質粒,采用pull down技術和Western Blot方法分析異源表達RIG-Ⅰ的泛素化水平,結果顯示,EV71病毒感染同樣顯著抑制細胞異源表達的RIG-Ⅰ泛素化。為了進一步確定RIG-Ⅰ泛素化的抑制類型,本研究將Flag-RIG-Ⅰ質粒分別與HA-Ub、HA-Ub K63或HA-Ub K48質粒共轉染至RD細胞,采用免疫共沉淀技術,分析RIG-Ⅰ的泛素化抑制類型。結果顯示,EV71病毒顯著抑制RIG-Ⅰ的K63位泛素化,而并不抑制K48位的泛素化。隨后,RD細胞共轉染HA-Ub和Flag-RIG-Ⅰ質粒,采用過表達技術提升RIG-Ⅰ的泛素化水平,并檢測IFN-β和ISGs mRNA的轉錄水平,結果顯示,上調RIG-Ⅰ的泛素化水平可以顯著增強IFN-β和ISGs的轉錄水平。上述研究結果說明,EV71病毒感染RD細胞之后,首先抑制宿主細胞RIG-Ⅰ受體K63位的泛素化,隨后阻斷IRF3的入核,抑制IFN-β的轉錄和表達,最終阻斷了天然免疫效應分子ISG54、ISG56的轉錄和表達,從而逃避了宿主天然免疫對自身的清除。本研究不僅有助于豐富EV71病毒與宿主天然免疫相互作用的理論知識,而且有助于發(fā)現(xiàn)潛在的抗病毒復制的新策略。
[Abstract]:Enterovirus 71 (Enteroviruses 71, EV71) belongs to the Picornaviridae family members, enterovirus, enterovirus A, is an important pathogen of hand foot and mouth disease in children. In recent years, the outbreak of EV71 virus in the world and popular, causing tens of thousands of people died after.EV71 virus invades a cell, the virus replication of the genome forming a double stranded RNA molecule, RIG- I will be recognition receptor pattern recognition of host cells, then RIG- receptor molecules in the ubiquitin ligase by ubiquitination, thereby initiating downstream of type I interferon signaling pathway. Our previous studies showed that EV71 infection can inhibit host RIG- receptor ubiquitination level. But I will eventually inhibit RIG- ubiquitination mediated by the EV71 virus to evade host innate immunity is unclear. Therefore, this paper will be to this discovery as the starting point, to clarify the EV71 virus inhibition of RIG- I pan In the escape this scientific problem of host innate immunity, EV71 virus to evade the host innate immune theory put forward new ideas. In order to study the replication of EV71 virus in RD cells, after EV71 infected RD cells 24 hours, using the optical microscope and fluorescence microscope at different time points to observe cell lesions. The results showed that EV71 virus infection 6 hours, detected expression of viral proteins, and cell lesions. Based on determining the infection time points, after the beta cells transfected with IFN- -Luc plasmid by dual luciferase reporter assay system for detection of EV71 virus infection promoter activity in RD cells. The results showed that IFN- beta, EV71 (MOI=20) the virus RD IFN- beta cells significantly reduced promoter activity. In order to confirm the inhibitory effect of EV71 virus on IFN- beta, RT-PCR method is used to influence the detection of viruses on IFN- beta transcription level. The results showed that Transcription, EV71 infection significantly inhibited the cell IFN-. Beta interferon regulated genes 3 (IRF3) into the nucleus is essential for the activation of host innate immunity, in order to confirm the possibility of EV71 virus through inhibition of IRF3 into the nucleus of antagonistic type I interferon pathway, RD cells transfected with pEGFP-IRF3 plasmid by immunofluorescence staining with IRF3 the nuclear situation. The results showed that EV71 infection inhibited the IRF3 into the nucleus. Interferon stimulated gene (ISG) is the end effector molecules exert antiviral effect, in order to study the effect of EV71 virus on ISGs, after EV71 infected RD cells, virus detection using RT-PCR ISG54 method, ISG56 and IFN- beta mRNA. The results showed that EV71 infection significantly reduced RD cell IFN- beta, ISGs mRNA transcription level. In order to study the effect of EV71 virus on RIG- receptor ubiquitination, EV71 infected RD cells, The ubiquitination level using the Western Blot method to detect endogenous RIG- 1. The results showed that EV71 infection significantly inhibited the endogenous RIG- of ubiquitination, and showed a time dependent manner. In order to confirm the inhibitory effect of EV71 virus on RIG- I, Flag-RIG- I and HA-Ub plasmid co transfection of RD cells, the ubiquitin level by pull down technology and Western Blot analysis showed that heterologous expression of RIG- 1, EV71 virus infection also significantly inhibited the heterologous expression of RIG- 1 ubiquitination. In order to further determine the type of inhibition of RIG- I ubiquitination, this research will Flag-RIG- 1 and HA-Ub HA-Ub plasmid respectively, K63 or HA-Ub K48 plasmid were transfected into RD cells. Using CO immunoprecipitation techniques, analysis of RIG- I ubiquitination inhibition type. The results showed that EV71 significantly inhibited the virus of RIG- I K63 ubiquitination, but does not inhibit K48 ubiquitination then, RD cells were transfected with HA-Ub plasmid and Flag-RIG- I, by over expression of ubiquitin to enhance the level of RIG- technology, and the results show that the detection level of transcription, IFN- beta and ISGs mRNA, the ubiquitin upregulation of RIG- I can significantly enhance the transcription level of IFN- beta and ISGs. The results show that after EV71 virus infection first of all RD cells, inhibit host cell receptor RIG- K63 ubiquitination, then blocking IRF3 into the nucleus, inhibition of transcription and expression of IFN- beta, eventually blocking the natural immune effector molecule ISG54, and the expression of ISG56 transcription, so as to avoid the removal of host innate immunity to itself. The research has not only theoretical knowledge enrich the EV71 virus and the host immune interaction, but also help to find new strategies for potential virus replication.

【學位授予單位】：黑龍江八一農(nóng)墾大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392

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