本文關鍵詞：高致病性病毒免疫拮抗蛋白的篩選　出處：《中國科學院大學(中國科學院武漢病毒研究所)》2017年碩士論文　論文類型：學位論文

  更多相關文章： 天然免疫 炎癥反應 高致病性病毒 I型干擾素 炎癥小體

【摘要】：天然免疫系統(tǒng)(innate immune system)在保護宿主抵御病原微生物感染過程中發(fā)揮著重要作用,其中模式識別受體(pattern recognition receptors,PRRs),即Toll樣受體(Toll-like receptors,TLRs)、RIG-I樣受體(RIG-I-like receptors,RLRs)和NOD樣受體(NOD-like receptors,NLRs)通過識別病原微生物的各類病原相關分子模式(pathogen-associated molecular patterns,PAMPs),誘導I型干擾素以及炎癥細胞因子的表達等來發(fā)揮宿主的抗病原微生物的免疫反應。炎癥反應是機體對抗病原微生物感染的一種病理過程,通過誘導一系列的炎癥細胞因子的表達,促進天然免疫與適應性免疫的發(fā)揮。病毒感染宿主細胞后會觸發(fā)宿主的抗病毒天然免疫反應,而病毒在長期進化過程中進化出各種逃逸宿主天然免疫的方式,通過逃逸宿主的天然免疫反應,完成病毒自身完整的生活周期。本研究中我們構建了5種高致病性病毒:拉沙熱病毒(Lassa fever virus)、埃博拉病毒(Ebola virus)、馬爾堡病毒(Marburg virus)、尼帕病毒(Nipah virus)和克里米亞剛果出血熱病毒(Crimean-congo hemorrhagic fever virus,CCHFV)的32種病毒蛋白過表達載體,并篩選了這些病毒蛋白對SeV誘導的Ⅰ型干擾素及炎癥細胞因子的活化、TNFα和IL-1β誘導的炎癥反應、IFNγ誘導的JAK-STAT活化和NLRP3炎癥小體的活化等信號通路的影響。通過篩選發(fā)現(xiàn)CCHFV蛋白的OTU domain,Nipah virus的P、W、V,以及Lassa virus的NP能夠顯著抑制SeV誘導的IFNβ、ISRE和NF-κB報告基因的激活。與報告基因試驗結果不同的是,Nipah virus的P、W、V在q-PCR實驗中對干擾素以及炎癥細胞因子的抑制作用不顯著;而Ebola virus的NP在q-PCR實驗中能夠明顯協(xié)同IFN(32)(16)的轉錄,而對下游的ISGs的轉錄沒有影響。通過檢測分泌到胞外的IFNb發(fā)現(xiàn),Ebola virus的NP能夠協(xié)同SeV誘導的IFNb的產(chǎn)生。另外,CCHFV的OTU domain和Nipah virus的P、W、V能夠抑制由IFNγ誘導的IRF1報告基因的活化。通過對病毒蛋白調控炎癥反應的篩選發(fā)現(xiàn),Nipah virus的C、P和V蛋白能夠抑制TNFα和IL-1β誘導的NF-κB的活化,Ebola virus的s GP能夠協(xié)同TNFα誘導的NF-κB的活化,而對IL-1β誘導的NF-κB的活化無顯著影響。在NLRP3炎癥小體活化方面,Nipah virus的C蛋白能夠顯著促進IL-1β的成熟和分泌。
[Abstract]:The innate immune system (immune) plays an important role in protecting the host against pathogenic microorganism infection. Pattern recognition receptor (PRRs). The RIG-I-like receptors of Toll like receptor is Toll-like receptor. RLRs) and NOD like receptor NOD-like receptors. NLRs by identifying pathogen-associated molecular patterns of various pathogen-associated microbes. PAMPsN induces the expression of interferon I and inflammatory cytokines to play an important role in the immune response of the host against pathogenic microbes. Inflammatory reaction is a pathological process of the body against the infection of pathogenic microorganisms. By inducing the expression of a series of inflammatory cytokines, we can promote innate immunity and adaptive immunity. Virus infection will trigger the innate immune response of the host. The virus evolved a variety of escape host innate immunity in the long-term evolution process, through the escape host innate immune response. In this study, we constructed five highly pathogenic viruses: Lassa fever virus. Ebola virus, Marburg virus. Nipah virus and Crimean-congo hemorrhagic fever virus. The activation of interferon type I and inflammatory cytokines induced by SeV was screened by 32 viral protein overexpression vectors of CCHFV. TNF 偽 and IL-1 尾 induced inflammation. IFN 緯 -induced activation of JAK-STAT and activation of NLRP3 inflammatory bodies. OTU domain of CCHFV protein was identified by screening. Nipah virus and Lassa virus NP could significantly inhibit SeV induced IFN 尾. The activation of ISRE and NF- 魏 B reporter genes was different from the results of reporter gene test. The inhibitory effect of V on interferon and inflammatory cytokines in q-PCR was not significant. However, the NP of Ebola virus could significantly cooperate with the transcription of IFNm32 (16) in q-PCR experiment. However, there was no effect on the transcription of downstream ISGs. By detecting the IFNb secreted to the extracellular, it was found that the NP of SeV virus could cooperate with the production of IFNb induced by SeV. The OTU domain of CCHFV and the OTU of Nipah virus. V can inhibit the activation of IRF1 reporter gene induced by IFN 緯. P and V protein could inhibit the activation of NF- 魏 B induced by TNF 偽 and IL-1 尾. The SGP of virus could cooperate with the activation of NF- 魏 B induced by TNF 偽. There was no significant effect on the activation of NF- 魏 B induced by IL-1 尾, but not on the activation of inflammatory bodies of NLRP3. C protein of Nipah virus can significantly promote the maturation and secretion of IL-1 尾.
【學位授予單位】：中國科學院大學(中國科學院武漢病毒研究所)
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392

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