本文選題：豬繁殖與呼吸綜合征病毒 + nsp4��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：豬繁殖與呼吸綜合征(porcine reproductive and respiratory syndrome,PRRS)是由豬繁殖與呼吸綜合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)引起的一種給全世界的養(yǎng)豬業(yè)造成巨大損失的病毒性傳染病。研究發(fā)現(xiàn)PRRSV通過自身編碼的蛋白抑制機體的天然免疫應(yīng)答,實現(xiàn)免疫逃避,但目前關(guān)于PRRSV編碼的nsp4蛋白抑制JAK-STAT信號通路的分子機制有待進一步研究。本研究通過研究PRRSV編碼的nsp4與JAK-STAT信號通路之間的相互作用,發(fā)現(xiàn)nsp4能切割關(guān)鍵信號分子STAT2。進一步分析了nsp4切割STAT2的氨基酸位點,探討nsp4切割STAT2在PRRSV調(diào)控天然免疫反應(yīng)中的作用。主要內(nèi)容如下:1.PRRSV感染細胞后抑制ISGs的表達通過相對熒光定量PCR檢測發(fā)現(xiàn)PRRSV感染PAMs細胞和MARC-145細胞后顯著抑制IFN-α誘導(dǎo)的2',5'-OAS,ISG15,ISG56和ISG60的m RNA水平;雙熒光素酶實驗結(jié)果顯示PRRSV感染MARC-145后可以顯著抑制IFN-α對ISRE-Luc的激活。為篩選出抑制ISRE活性的蛋白,將PRRSV編碼的所有結(jié)構(gòu)蛋白和非結(jié)構(gòu)蛋白的真核表達質(zhì)粒與熒光素酶報告質(zhì)粒ISRE-Luc和p GL-4.74共轉(zhuǎn)染HEK-293T細胞,雙熒光素酶實驗結(jié)果顯示PRRSV的非結(jié)構(gòu)蛋白nsp1a、nsp1b、nsp4、nsp11、nsp12,結(jié)構(gòu)蛋白GP3和N均能抑制IFN-α誘導(dǎo)的ISRE啟動子活性,其中nsp4抑制效果顯著。2.PRRSV編碼的nsp4蛋白依賴其絲氨酸蛋白酶活性抑制IFN-α誘導(dǎo)的ISRE活性將真核表達質(zhì)粒nsp4進行倍比稀釋與熒光素酶報告質(zhì)粒ISRE-Luc共轉(zhuǎn)染HEK-293T或3D4細胞,雙熒光素酶檢測結(jié)果顯示在這兩種細胞上nsp4均能顯著抑制IFN-α誘導(dǎo)的ISRE啟動子活性,并且呈明顯的劑量依賴;利用雙熒光素酶報告系統(tǒng)證明nsp4抑制IFN-α誘導(dǎo)的ISRE啟動子活性依賴其絲氨酸蛋白酶活性。3.PRRSV編碼的nsp4蛋白切割STAT2位點為E719本研究發(fā)現(xiàn)超表達nsp4對STAT2蛋白的m RNA水平?jīng)]有影響,而切割JAK-STAT信號通路中關(guān)鍵蛋白STAT2,且呈明顯的劑量依賴;本研究證明nsp4切割STAT2不依賴泛素-蛋白酶體途徑和細胞凋亡途徑,而是依賴于自身的絲氨酸蛋白酶活性,并鑒定出nsp4切割STAT2的位點為E719。PRRSV感染MARC-145細胞后,Western Blot檢測發(fā)現(xiàn)內(nèi)源性STAT2被降解,但是檢測不到切割條帶。4.PRRSV編碼的nsp4蛋白拮抗JAK-STAT信號通路本研究將nsp4真核表達質(zhì)粒與STAT1和STAT2真核表達質(zhì)粒共轉(zhuǎn)染HEK-293T細胞,通過Co-IP實驗發(fā)現(xiàn)PRRSV編碼的nsp4蛋白切割STAT2之后,STAT1和STAT2的相互作用顯著減弱。將STAT2全長或截短突變體與STAT1和IRF9共轉(zhuǎn)染,雙熒光素酶實驗和熒光定量PCR結(jié)果顯示只有全長STAT2可以與STAT1和IRF9作用激活I(lǐng)SRE,誘導(dǎo)ISGs表達,截短的STAT2不能激活I(lǐng)SRE活性和ISGs的表達,nsp4切割STAT2抑制了JAK-STAT信號通路的信號傳遞。綜上所述,本研究首次揭示了PRRSV編碼的nsp4蛋白在拮抗JAK-STAT信號通路中的重要作用;發(fā)現(xiàn)PRRSV編碼的nsp4蛋白依賴其自身的絲氨酸蛋白酶活性切割JAK-STAT信號通路中重要信號分子STAT2蛋白,阻止信號向下游傳遞,抑制ISRE啟動子活性,以及ISGs的轉(zhuǎn)錄表達。同時鑒定出nsp4切割STAT2的位點是E719。本研究加深了我們對PRRSV致病和免疫逃避機制的理解,為PRRSV疫苗研究與開發(fā)提供理論依據(jù)。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRS) is a viral infection caused by the porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus) causing huge losses to the world's pig industry. White inhibits the natural immune response of the body and realizes immune escape, but the molecular mechanism of the inhibition of the JAK-STAT signaling pathway by the PRRSV encoded NSP4 protein remains to be further studied. This study finds that the NSP4 can cut the key signal molecule STAT2. further by studying the interaction between the PRRSV encoded NSP4 and the JAK-STAT signaling pathway. The amino acid site of NSP4 cutting STAT2 was analyzed, and the role of NSP4 cutting STAT2 in PRRSV regulation of natural immune response was discussed. The main contents are as follows: the expression of the inhibition of ISGs in 1.PRRSV infected cells by relative fluorescence quantitative PCR detection shows that PRRSV infected PAMs cells and MARC-145 cells obviously inhibit IFN- alpha induction. The m RNA level of G60, and the results of double luciferase test showed that PRRSV could significantly inhibit the activation of ISRE-Luc after MARC-145 infection. In order to screen out the protein to inhibit ISRE activity, all the structural proteins and the eukaryotic expression plasmids of the PRRSV encoded protein and the luciferase reporter plasmid ISRE-Luc and P GL-4.74 were co transfected. The results of the double luciferase experiment showed that the non structural protein nsp1a, nsp1b, NSP4, nsp11, nsp12, structural protein GP3 and N all inhibited the IFN- alpha induced ISRE promoter activity, and the NSP4 inhibition effect was significantly dependent on the inhibitory activity of the serine protease. HEK-293T or 3D4 cells were co transfected with doubling dilution and luciferase reporter plasmid ISRE-Luc. The results of double luciferase detection showed that NSP4 could significantly inhibit ISRE promoter activity induced by IFN- A and showed a significant dose dependence. The dual luciferase reporter system demonstrated that NSP4 inhibited IFN- alpha induced ISRE initiation. The subactivity dependent on the NSP4 protein cut STAT2 loci encoded by the serine protease activity.3.PRRSV was E719. The study found that the overexpression NSP4 had no effect on the m RNA level of STAT2 protein, while the key protein STAT2 in the JAK-STAT signaling pathway was significantly dose dependent. This study proved that NSP4 cleavage STAT2 did not depend on the ubiquitin proteasome. The pathway and apoptotic pathway are dependent on the serine protease activity of its own, and it is identified that NSP4 STAT2 cleavage site is E719.PRRSV infected MARC-145 cells. Western Blot detection found that endogenous STAT2 is degraded, but the NSP4 protein antagonistic JAK-STAT signaling pathway of the cleavage band.4.PRRSV is not detected for NSP4. The eukaryotic expression plasmid co transfected HEK-293T cells with STAT1 and STAT2 eukaryotic expression plasmids. The interaction between STAT1 and STAT2 decreased significantly after PRRSV encoded NSP4 protein cleavage STAT2 by Co-IP experiment. The total length or truncated mutant of STAT2 was co transfected with STAT1 and IRF9. The results of double fluorescent enzyme experiment and fluorescence quantitative determination showed that only whole Long STAT2 can activate ISRE and induce ISGs expression with STAT1 and IRF9. The truncated STAT2 can not activate the ISRE activity and the expression of ISGs. NSP4 cutting STAT2 inhibits the signal transmission of JAK-STAT signaling pathway. The NSP4 protein of the code relies on its own serine protease activity to cut the important signal molecule STAT2 protein in the JAK-STAT signaling pathway, preventing the signal from passing downstream, inhibiting the activity of the ISRE promoter, and the transcriptional expression of ISGs. At the same time, the site of NSP4 cutting STAT2 is E719. Ben research deepened our pathogenesis and immune escape to PRRSV. The understanding of the mechanism provides a theoretical basis for the research and development of PRRSV vaccine.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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