IBDV基因組dsRNA與宿主細(xì)胞RNA結(jié)合蛋白Staufen1互作調(diào)控IBDV復(fù)制及其機(jī)制
發(fā)布時(shí)間：2019-04-15 23:18
【摘要】：IBDV屬于雙RNA病毒科,禽雙RNA病毒屬,是傳染性法氏囊病(IBD)的病原體。IBD是一種急性、高度傳染性疾病,可以導(dǎo)致青年雞法氏囊出現(xiàn)炎癥、萎縮,還會(huì)引起嚴(yán)重的免疫抑制。宿主細(xì)胞蛋白Staufen1(Stau1)屬于雙鏈RNA結(jié)合蛋白家族的一員,參與mRNA的轉(zhuǎn)運(yùn)、翻譯以及降解。除此之外,還參與某些RNA病毒的生命周期。本文研究了Stau1蛋白與非己的IBDV基因組dsRNA相互作用調(diào)控IBDV的復(fù)制,并探究了其分子機(jī)制。為了研究雞源Stau1蛋白與IBDV基因組dsRNA之間的關(guān)系,本研究首先克隆了雞Stau1基因,構(gòu)建真核表達(dá)載體以及相關(guān)的原核表達(dá)載體。本文采用IBDV dsRNA pull-down實(shí)驗(yàn)、凝膠遷移實(shí)驗(yàn)以及質(zhì)譜鑒定來研究Stau1蛋白與IBDV基因組dsRNA是否相互作用以及Stau1與dsRNA結(jié)合的具體結(jié)構(gòu)區(qū)域。利用間接免疫熒光反應(yīng)探究了Stau1蛋白與IBDV基因組dsRNA在細(xì)胞中的共定位情況。我們通過建立IBDV細(xì)胞感染模型,利用RNA干擾技術(shù)、蝕斑實(shí)驗(yàn)以及RT-PCR實(shí)驗(yàn)研究了Stau1蛋白對(duì)IBDV復(fù)制的影響。另外利用熒光素酶報(bào)告基因系統(tǒng),研究了Stau1對(duì)IBDV基因組dsRNA誘導(dǎo)產(chǎn)生IFN-β的影響。采用IBDV基因組dsRNA pull-down實(shí)驗(yàn),比較了VP3、MDA5、Stau1與IBDV基因組dsRNA的親和力。結(jié)果:(1)無論在體外還是在體內(nèi),雞源Stau1蛋白都可以直接與IBDV基因組dsRNA相互結(jié)合,并在細(xì)胞中發(fā)生共定位,且N-端1-468個(gè)氨基酸負(fù)責(zé)Stau1結(jié)合dsRNA的功能;(2)下調(diào)Stau1蛋白的表達(dá),可以抑制后期IBDV病毒復(fù)制,而Stau1上調(diào)表達(dá)則可以促進(jìn)IBDV病毒復(fù)制;(3)下調(diào)Stau1表達(dá)會(huì)選擇性的促進(jìn)IBDV dsRNA誘導(dǎo)產(chǎn)生的IFN-β的表達(dá),而對(duì)IFN-α沒有影響;(4)Stau1可以通過結(jié)合IBDV dsRNA來抑制感染IBDV誘導(dǎo)的IFN-β的產(chǎn)生;(5)Stau1與MDA5競(jìng)爭性結(jié)合IBDV dsRNA,來抑制IBDV誘導(dǎo)產(chǎn)生的IFN-β,并且病毒蛋白VP3與雞Stau1協(xié)同發(fā)揮抑制IFN-β產(chǎn)生的作用。結(jié)論:本研究首次發(fā)現(xiàn)雞Stau1蛋白可以與IBDV dsRNA相互作用,并可以通過與MDA5競(jìng)爭結(jié)合dsRNA,來抑制IBDV誘導(dǎo)產(chǎn)生的IFN-β,并且病毒蛋白VP3與雞Stau1協(xié)同發(fā)揮抑制IFN-β產(chǎn)生的作用,從而達(dá)到促進(jìn)IBDV復(fù)制的目的。本次研究促進(jìn)我們對(duì)IBDV致病和免疫機(jī)制的認(rèn)知,為防控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- 偽. (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.
【學(xué)位授予單位】：浙江農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65
本文編號(hào)：2458585
[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- 偽. (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.
【學(xué)位授予單位】：浙江農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 周宗安,王永山,鄧小昭,刁振宇,高建,施正良,羅函祿,方元;傳染性法氏囊病病毒的生態(tài)學(xué)與流行病學(xué)研究[J];中國獸醫(yī)學(xué)報(bào);1998年05期
，本文編號(hào)：2458585
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