猪繁殖与呼吸综合征病毒NSP4切割STAT2分子机制的研究

发布时间：2018-05-15 02:12

  本文选题：猪繁殖与呼吸综合征病毒 + nsp4　； 参考：《华中农业大学》2017年硕士论文

【摘要】：猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)引起的一种给全世界的养猪业造成巨大损失的病毒性传染病。研究发现PRRSV通过自身编码的蛋白抑制机体的天然免疫应答,实现免疫逃避,但目前关于PRRSV编码的nsp4蛋白抑制JAK-STAT信号通路的分子机制有待进一步研究。本研究通过研究PRRSV编码的nsp4与JAK-STAT信号通路之间的相互作用,发现nsp4能切割关键信号分子STAT2。进一步分析了nsp4切割STAT2的氨基酸位点,探讨nsp4切割STAT2在PRRSV调控天然免疫反应中的作用。主要内容如下:1.PRRSV感染细胞后抑制ISGs的表达通过相对荧光定量PCR检测发现PRRSV感染PAMs细胞和MARC-145细胞后显著抑制IFN-α诱导的2',5'-OAS,ISG15,ISG56和ISG60的m RNA水平;双荧光素酶实验结果显示PRRSV感染MARC-145后可以显著抑制IFN-α对ISRE-Luc的激活。为筛选出抑制ISRE活性的蛋白,将PRRSV编码的所有结构蛋白和非结构蛋白的真核表达质粒与荧光素酶报告质粒ISRE-Luc和p GL-4.74共转染HEK-293T细胞,双荧光素酶实验结果显示PRRSV的非结构蛋白nsp1a、nsp1b、nsp4、nsp11、nsp12,结构蛋白GP3和N均能抑制IFN-α诱导的ISRE启动子活性,其中nsp4抑制效果显著。2.PRRSV编码的nsp4蛋白依赖其丝氨酸蛋白酶活性抑制IFN-α诱导的ISRE活性将真核表达质粒nsp4进行倍比稀释与荧光素酶报告质粒ISRE-Luc共转染HEK-293T或3D4细胞,双荧光素酶检测结果显示在这两种细胞上nsp4均能显著抑制IFN-α诱导的ISRE启动子活性,并且呈明显的剂量依赖;利用双荧光素酶报告系统证明nsp4抑制IFN-α诱导的ISRE启动子活性依赖其丝氨酸蛋白酶活性。3.PRRSV编码的nsp4蛋白切割STAT2位点为E719本研究发现超表达nsp4对STAT2蛋白的m RNA水平没有影响,而切割JAK-STAT信号通路中关键蛋白STAT2,且呈明显的剂量依赖;本研究证明nsp4切割STAT2不依赖泛素-蛋白酶体途径和细胞凋亡途径,而是依赖于自身的丝氨酸蛋白酶活性,并鉴定出nsp4切割STAT2的位点为E719。PRRSV感染MARC-145细胞后,Western Blot检测发现内源性STAT2被降解,但是检测不到切割条带。4.PRRSV编码的nsp4蛋白拮抗JAK-STAT信号通路本研究将nsp4真核表达质粒与STAT1和STAT2真核表达质粒共转染HEK-293T细胞,通过Co-IP实验发现PRRSV编码的nsp4蛋白切割STAT2之后,STAT1和STAT2的相互作用显著减弱。将STAT2全长或截短突变体与STAT1和IRF9共转染,双荧光素酶实验和荧光定量PCR结果显示只有全长STAT2可以与STAT1和IRF9作用激活ISRE,诱导ISGs表达,截短的STAT2不能激活ISRE活性和ISGs的表达,nsp4切割STAT2抑制了JAK-STAT信号通路的信号传递。综上所述,本研究首次揭示了PRRSV编码的nsp4蛋白在拮抗JAK-STAT信号通路中的重要作用;发现PRRSV编码的nsp4蛋白依赖其自身的丝氨酸蛋白酶活性切割JAK-STAT信号通路中重要信号分子STAT2蛋白,阻止信号向下游传递,抑制ISRE启动子活性,以及ISGs的转录表达。同时鉴定出nsp4切割STAT2的位点是E719。本研究加深了我们对PRRSV致病和免疫逃避机制的理解,为PRRSV疫苗研究与开发提供理论依据。
[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.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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