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

  更多相關(guān)文章： 天然免疫 炎癥反應(yīng) 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(hù)宿主抵御病原微生物感染過(guò)程中發(fā)揮著重要作用,其中模式識(shí)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(guò)識(shí)別病原微生物的各類病原相關(guān)分子模式(pathogen-associated molecular patterns,PAMPs),誘導(dǎo)I型干擾素以及炎癥細(xì)胞因子的表達(dá)等來(lái)發(fā)揮宿主的抗病原微生物的免疫反應(yīng)。炎癥反應(yīng)是機(jī)體對(duì)抗病原微生物感染的一種病理過(guò)程,通過(guò)誘導(dǎo)一系列的炎癥細(xì)胞因子的表達(dá),促進(jìn)天然免疫與適應(yīng)性免疫的發(fā)揮。病毒感染宿主細(xì)胞后會(huì)觸發(fā)宿主的抗病毒天然免疫反應(yīng),而病毒在長(zhǎng)期進(jìn)化過(guò)程中進(jìn)化出各種逃逸宿主天然免疫的方式,通過(guò)逃逸宿主的天然免疫反應(yīng),完成病毒自身完整的生活周期。本研究中我們構(gòu)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(guò)表達(dá)載體,并篩選了這些病毒蛋白對(duì)SeV誘導(dǎo)的Ⅰ型干擾素及炎癥細(xì)胞因子的活化、TNFα和IL-1β誘導(dǎo)的炎癥反應(yīng)、IFNγ誘導(dǎo)的JAK-STAT活化和NLRP3炎癥小體的活化等信號(hào)通路的影響。通過(guò)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(dǎo)的IFNβ、ISRE和NF-κB報(bào)告基因的激活。與報(bào)告基因試驗(yàn)結(jié)果不同的是,Nipah virus的P、W、V在q-PCR實(shí)驗(yàn)中對(duì)干擾素以及炎癥細(xì)胞因子的抑制作用不顯著;而Ebola virus的NP在q-PCR實(shí)驗(yàn)中能夠明顯協(xié)同IFN(32)(16)的轉(zhuǎn)錄,而對(duì)下游的ISGs的轉(zhuǎn)錄沒有影響。通過(guò)檢測(cè)分泌到胞外的IFNb發(fā)現(xiàn),Ebola virus的NP能夠協(xié)同SeV誘導(dǎo)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(dǎo)的IRF1報(bào)告基因的活化。通過(guò)對(duì)病毒蛋白調(diào)控炎癥反應(yīng)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(dǎo)的NF-κB的活化,Ebola virus的s GP能夠協(xié)同TNFα誘導(dǎo)的NF-κB的活化,而對(duì)IL-1β誘導(dǎo)的NF-κB的活化無(wú)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(jìn)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(xué)位授予單位】：中國(guó)科學(xué)院大學(xué)(中國(guó)科學(xué)院武漢病毒研究所)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392

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