高致病性病毒免疫拮抗蛋白的筛选

发布时间：2018-01-06 19:33

本文关键词：高致病性病毒免疫拮抗蛋白的筛选　出处：《中国科学院大学(中国科学院武汉病毒研究所)》2017年硕士论文　论文类型：学位论文

【摘要】：天然免疫系统(innate immune system)在保护宿主抵御病原微生物感染过程中发挥着重要作用,其中模式识别受体(pattern recognition receptors,PRRs),即Toll样受体(Toll-like receptors,TLRs)、RIG-I样受体(RIG-I-like receptors,RLRs)和NOD样受体(NOD-like receptors,NLRs)通过识别病原微生物的各类病原相关分子模式(pathogen-associated molecular patterns,PAMPs),诱导I型干扰素以及炎症细胞因子的表达等来发挥宿主的抗病原微生物的免疫反应。炎症反应是机体对抗病原微生物感染的一种病理过程,通过诱导一系列的炎症细胞因子的表达,促进天然免疫与适应性免疫的发挥。病毒感染宿主细胞后会触发宿主的抗病毒天然免疫反应,而病毒在长期进化过程中进化出各种逃逸宿主天然免疫的方式,通过逃逸宿主的天然免疫反应,完成病毒自身完整的生活周期。本研究中我们构建了5种高致病性病毒:拉沙热病毒(Lassa fever virus)、埃博拉病毒(Ebola virus)、马尔堡病毒(Marburg virus)、尼帕病毒(Nipah virus)和克里米亚刚果出血热病毒(Crimean-congo hemorrhagic fever virus,CCHFV)的32种病毒蛋白过表达载体,并筛选了这些病毒蛋白对SeV诱导的Ⅰ型干扰素及炎症细胞因子的活化、TNFα和IL-1β诱导的炎症反应、IFNγ诱导的JAK-STAT活化和NLRP3炎症小体的活化等信号通路的影响。通过筛选发现CCHFV蛋白的OTU domain,Nipah virus的P、W、V,以及Lassa virus的NP能够显著抑制SeV诱导的IFNβ、ISRE和NF-κB报告基因的激活。与报告基因试验结果不同的是,Nipah virus的P、W、V在q-PCR实验中对干扰素以及炎症细胞因子的抑制作用不显著;而Ebola virus的NP在q-PCR实验中能够明显协同IFN(32)(16)的转录,而对下游的ISGs的转录没有影响。通过检测分泌到胞外的IFNb发现,Ebola virus的NP能够协同SeV诱导的IFNb的产生。另外,CCHFV的OTU domain和Nipah virus的P、W、V能够抑制由IFNγ诱导的IRF1报告基因的活化。通过对病毒蛋白调控炎症反应的筛选发现,Nipah virus的C、P和V蛋白能够抑制TNFα和IL-1β诱导的NF-κB的活化,Ebola virus的s GP能够协同TNFα诱导的NF-κB的活化,而对IL-1β诱导的NF-κB的活化无显著影响。在NLRP3炎症小体活化方面,Nipah virus的C蛋白能够显著促进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 伪 and IL-1 尾 induced inflammation. IFN 纬 -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 纬. P and V protein could inhibit the activation of NF- 魏 B induced by TNF 伪 and IL-1 尾. The SGP of virus could cooperate with the activation of NF- 魏 B induced by TNF 伪. 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.
【学位授予单位】：中国科学院大学(中国科学院武汉病毒研究所)
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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