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IBDV基因组dsRNA与宿主细胞RNA结合蛋白Staufen1互作调控IBDV复制及其机制

发布时间:2019-04-15 23:18
【摘要】:IBDV属于双RNA病毒科,禽双RNA病毒属,是传染性法氏囊病(IBD)的病原体。IBD是一种急性、高度传染性疾病,可以导致青年鸡法氏囊出现炎症、萎缩,还会引起严重的免疫抑制。宿主细胞蛋白Staufen1(Stau1)属于双链RNA结合蛋白家族的一员,参与mRNA的转运、翻译以及降解。除此之外,还参与某些RNA病毒的生命周期。本文研究了Stau1蛋白与非己的IBDV基因组dsRNA相互作用调控IBDV的复制,并探究了其分子机制。为了研究鸡源Stau1蛋白与IBDV基因组dsRNA之间的关系,本研究首先克隆了鸡Stau1基因,构建真核表达载体以及相关的原核表达载体。本文采用IBDV dsRNA pull-down实验、凝胶迁移实验以及质谱鉴定来研究Stau1蛋白与IBDV基因组dsRNA是否相互作用以及Stau1与dsRNA结合的具体结构区域。利用间接免疫荧光反应探究了Stau1蛋白与IBDV基因组dsRNA在细胞中的共定位情况。我们通过建立IBDV细胞感染模型,利用RNA干扰技术、蚀斑实验以及RT-PCR实验研究了Stau1蛋白对IBDV复制的影响。另外利用荧光素酶报告基因系统,研究了Stau1对IBDV基因组dsRNA诱导产生IFN-β的影响。采用IBDV基因组dsRNA pull-down实验,比较了VP3、MDA5、Stau1与IBDV基因组dsRNA的亲和力。结果:(1)无论在体外还是在体内,鸡源Stau1蛋白都可以直接与IBDV基因组dsRNA相互结合,并在细胞中发生共定位,且N-端1-468个氨基酸负责Stau1结合dsRNA的功能;(2)下调Stau1蛋白的表达,可以抑制后期IBDV病毒复制,而Stau1上调表达则可以促进IBDV病毒复制;(3)下调Stau1表达会选择性的促进IBDV dsRNA诱导产生的IFN-β的表达,而对IFN-α没有影响;(4)Stau1可以通过结合IBDV dsRNA来抑制感染IBDV诱导的IFN-β的产生;(5)Stau1与MDA5竞争性结合IBDV dsRNA,来抑制IBDV诱导产生的IFN-β,并且病毒蛋白VP3与鸡Stau1协同发挥抑制IFN-β产生的作用。结论:本研究首次发现鸡Stau1蛋白可以与IBDV dsRNA相互作用,并可以通过与MDA5竞争结合dsRNA,来抑制IBDV诱导产生的IFN-β,并且病毒蛋白VP3与鸡Stau1协同发挥抑制IFN-β产生的作用,从而达到促进IBDV复制的目的。本次研究促进我们对IBDV致病和免疫机制的认知,为防控IBD提供新的思路。
[Abstract]:IBDV is an acute and highly infectious disease, which can cause inflammation, atrophy and severe immunosuppression of bursa Fabricius in young chickens. Host cell protein Staufen1 (Stau1), a member of the double-stranded RNA binding protein family, is involved in the transport, translation and degradation of mRNA. In addition, it is involved in the life cycle of some RNA viruses. In this paper, we studied the interaction between Stau1 protein and non-IBDV genomic dsRNA to regulate the replication of IBDV and explore its molecular mechanism. In order to study the relationship between chicken-derived Stau1 protein and IBDV genomic dsRNA, chicken Stau1 gene was cloned, eukaryotic expression vector and related prokaryotic expression vector were constructed. In this paper, IBDV dsRNA pull-down assay, gel migration test and mass spectrometry were used to study the interaction between Stau1 protein and IBDV genomic dsRNA and the specific structural domain of Stau1 binding to dsRNA. The co-localization of Stau1 protein and IBDV genomic dsRNA in cells was investigated by indirect immunofluorescence reaction. We established IBDV cell infection model and studied the effect of Stau1 protein on IBDV replication by using RNA interference technique, plaque assay and RT-PCR assay. In addition, luciferase reporter gene system was used to study the effect of Stau1 on IFN- 尾 induced by IBDV genomic dsRNA. IBDV genomic dsRNA pull-down assay was used to compare the affinity between VP3,MDA5,Stau1 and IBDV genomic dsRNA. Results: (1) both in vitro and in vivo, chicken-derived Stau1 protein could interact directly with IBDV genomic dsRNA and co-localize in cells. The N-terminal 1, 468 amino acids were responsible for the function of Stau1 binding to dsRNA. (2) the down-regulation of the expression of Stau1 protein could inhibit the replication of IBDV virus in the late stage, while the up-regulation of the expression of Stau1 could promote the replication of IBDV virus. (3) down-regulation of the expression of Stau1 could selectively promote the expression of IFN- 尾 induced by IBDV dsRNA, but had no effect on IFN- 伪. (4) Stau1 could inhibit the production of IFN- 尾 induced by infection with IBDV by binding IBDV dsRNA. (5) Stau1 and MDA5 compete with IBDV dsRNA, to inhibit the production of IFN- 尾 induced by IBDV, and the viral protein VP3 and chicken Stau1 play a synergistic role in inhibiting the production of IFN- 尾. Conclusion: it was found for the first time that chicken Stau1 protein could interact with IBDV dsRNA and inhibit IBDV-induced IFN- 尾 by competing with MDA5 and binding dsRNA, and virus protein VP3 and chicken Stau1 could inhibit IFN- 尾 production. In order to achieve the purpose of promoting IBDV replication. This study promotes our understanding of the pathogenesis and immune mechanism of IBDV and provides new ideas for prevention and control of IBD.
【学位授予单位】:浙江农林大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:S852.65

【参考文献】

相关期刊论文 前1条

1 周宗安,王永山,邓小昭,刁振宇,高建,施正良,罗函禄,方元;传染性法氏囊病病毒的生态学与流行病学研究[J];中国兽医学报;1998年05期



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