口蹄疫病毒2B蛋白拮抗RIG-I抗病毒作用研究
发布时间：2018-10-14 18:13
【摘要】：口蹄疫病毒所引起的口蹄疫是偶蹄动物的烈性传染病,世界动物卫生组织(OIE)一直将其列为法定必报病,在我国被农业部列为一类传染病之首,口蹄疫的暴发往往会导致严重的经济损失。RIG-I样受体是天然免疫系统中一类重要的模式识别受体,能够识别细胞质中的病毒RNA。RLR信号通路既受到宿主的严格调控,也能够作为病毒逃逸宿主抗病毒反应的靶点。目前,很多RNA病毒拮抗RIG-I抗病毒天然免疫的分子机制已经研究清楚,而口蹄疫病毒对RIG-I抗病毒作用的拮抗机制研究尚不明朗。本研究以口蹄疫病毒拮抗RIG-I抗病毒天然免疫为切入点,构建了猪RIG-I以及A型口蹄疫病毒蛋白(VP1、VP2、VP3、VP4、2B、3A、3B、3C、3D)的真核表达质粒,并通过western blotting和间接免疫荧光实验对构建的真核表达质粒进行了表达验证,证明其成功表达,同时证明猪RIG-I蛋白定位于细胞的胞浆中。感染实验发现口蹄疫病毒能够诱导细胞内RIG-I的转录上调,表明两者间存在着重要联系。过表达实验证实RIG-I具有抑制FMDV复制的作用,而下调表达RIG-I可以促进FMDV的复制,表明RIG-I在机体抗口蹄疫病毒感染过程中发挥着重要的作用。同时,在口蹄疫病毒感染过程中,还发现RIG-I在蛋白水平上随感染时间的延长会被逐渐降解。随后将构建好的口蹄疫病毒各个病毒蛋白质粒分别与RIG-I质粒共转染293-T细胞,发现口蹄疫病毒2B蛋白可以降解RIG-I,而且通过western blotting和间接免疫荧光实验证实2B蛋白可以剂量依赖性的降解RIG-I。为了确定2B蛋白与RIG-I互作的结合域,本研究将2B基因分为三段分别构建了突变体质粒,命名为2B(1-50)、2B(50-100)、2B(100-150),接着将2B各个突变体质粒分别与RIG-I质粒共转染293-T细胞,发现2B(100-150)即2B羧基端可以降解RIG-I。然后又对2B和内源性RIG-I的关系进行了研究,结果发现2B蛋白可以剂量依赖性的抑制内源性RIG-I的转录和表达,进一步开展了2B及其突变体过表达实验,结果证实2B(1-50)即2B氨基端可以抑制RIG-I mRNA的转录。为了探明2B是通过与RIG-I直接互作还是通过其他途径来进行降解RIG-I的分子机制,笔者又做了间接免疫荧光、免疫共沉淀以及蛋白酶体和溶酶体抑制实验,结果显示2B和RIG-I存在共定位并发生互作,并且2B降解RIG-I不利用蛋白酶体和溶酶体途径,而是通过与RIG-I直接互作发挥降解作用。总之,本研究证实了猪RIG-I具有抗口蹄疫病毒感染的功能,并且发现口蹄疫病毒2B蛋白具有拮抗猪RIG-I抗病毒天然免疫的作用,初步探明了2B蛋白降解RIG-I的分子机制。这些研究结果,揭示了口蹄疫病毒免疫逃逸的机制,为进一步研究宿主与病原间相互作用的分子机制以及开发抗病毒药物和研制新型疫苗提供了理论依据。
[Abstract]:Foot-and-mouth disease caused by foot-and-mouth disease virus is a severe infectious disease of cloven-hoofed animals. The (OIE) of the World Organization of Animal Health has always listed it as a mandatory disease, and has been listed by the Ministry of Agriculture as one of the first class infectious diseases in China. The outbreak of foot-and-mouth disease often results in serious economic losses. RIG-I like receptors are important pattern recognition receptors in the innate immune system, which can recognize the viral RNA.RLR signaling pathway in the cytoplasm, which is strictly regulated by the host. It can also be used as a target for virus escape host anti-virus response. At present, many molecular mechanisms of RNA virus antagonizing RIG-I anti-virus innate immunity have been studied, but the mechanism of FMDV antagonistic effect on RIG-I is still unclear. In this study, the eukaryotic expression plasmids of porcine RIG-I and type A foot-and-mouth disease virus (VP1,VP2,VP3,VP4,2B,3A,3B,3C,3D) protein were constructed by using foot-and-mouth disease virus (FMDV) antagonism against RIG-I innate immunity. The expression of the constructed eukaryotic expression plasmid was verified by western blotting and indirect immunofluorescence assay. It was proved that the expression of porcine RIG-I protein was located in the cytoplasm of the cells. It was found that foot-and-mouth disease virus could induce up-regulation of RIG-I transcription in cells, indicating that there was an important relationship between the two. Overexpression experiments confirmed that RIG-I could inhibit FMDV replication, while down-regulation of RIG-I could promote FMDV replication, suggesting that RIG-I plays an important role in the process of anti-foot-and-mouth disease virus infection. At the same time, in the process of FMDV infection, the protein level of RIG-I was gradually degraded with the time of infection. Then, the constructed FMDV proteins were co-transfected with RIG-I plasmid into 293-T cells. It was found that foot-and-mouth disease virus 2B protein could degrade RIG-I, and western blotting and indirect immunofluorescence assay confirmed that 2B protein could degrade RIG-I. in a dose-dependent manner. In order to determine the binding domain between 2B protein and RIG-I, we divided 2B gene into three segments, named 2B (1-50), 2B (50-100), 2B (100-150), and cotransfected 293-T cells with RIG-I plasmid. It was found that 2B (100-150), that is, 2B carboxyl terminal, can degrade RIG-I.. Then the relationship between 2B and endogenous RIG-I was studied. The results showed that 2B protein could inhibit the transcription and expression of endogenous RIG-I in a dose-dependent manner. The results showed that 2B (1-50)-2B amino terminal could inhibit the transcription of RIG-I mRNA. In order to understand the molecular mechanism of RIG-I degradation by direct interaction with RIG-I or by other means, indirect immunofluorescence, immunoprecipitation, proteasome and lysosome inhibition were also performed. The results showed that there was colocalization and interaction between 2B and RIG-I, and 2B degradation of RIG-I did not utilize the proteasome and lysosome pathway, but played a direct role in degradation by direct interaction with RIG-I. In conclusion, this study confirmed that porcine RIG-I had the function of resisting foot-and-mouth disease virus infection, and found that foot-and-mouth disease virus 2B protein could antagonize the innate immunity of porcine RIG-I against virus infection. The molecular mechanism of RIG-I degradation by 2B protein was preliminarily demonstrated. These results reveal the mechanism of immune escape of foot-and-mouth disease virus and provide a theoretical basis for further study on the molecular mechanism of host-pathogen interaction and the development of antiviral drugs and new vaccines.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
[Abstract]:Foot-and-mouth disease caused by foot-and-mouth disease virus is a severe infectious disease of cloven-hoofed animals. The (OIE) of the World Organization of Animal Health has always listed it as a mandatory disease, and has been listed by the Ministry of Agriculture as one of the first class infectious diseases in China. The outbreak of foot-and-mouth disease often results in serious economic losses. RIG-I like receptors are important pattern recognition receptors in the innate immune system, which can recognize the viral RNA.RLR signaling pathway in the cytoplasm, which is strictly regulated by the host. It can also be used as a target for virus escape host anti-virus response. At present, many molecular mechanisms of RNA virus antagonizing RIG-I anti-virus innate immunity have been studied, but the mechanism of FMDV antagonistic effect on RIG-I is still unclear. In this study, the eukaryotic expression plasmids of porcine RIG-I and type A foot-and-mouth disease virus (VP1,VP2,VP3,VP4,2B,3A,3B,3C,3D) protein were constructed by using foot-and-mouth disease virus (FMDV) antagonism against RIG-I innate immunity. The expression of the constructed eukaryotic expression plasmid was verified by western blotting and indirect immunofluorescence assay. It was proved that the expression of porcine RIG-I protein was located in the cytoplasm of the cells. It was found that foot-and-mouth disease virus could induce up-regulation of RIG-I transcription in cells, indicating that there was an important relationship between the two. Overexpression experiments confirmed that RIG-I could inhibit FMDV replication, while down-regulation of RIG-I could promote FMDV replication, suggesting that RIG-I plays an important role in the process of anti-foot-and-mouth disease virus infection. At the same time, in the process of FMDV infection, the protein level of RIG-I was gradually degraded with the time of infection. Then, the constructed FMDV proteins were co-transfected with RIG-I plasmid into 293-T cells. It was found that foot-and-mouth disease virus 2B protein could degrade RIG-I, and western blotting and indirect immunofluorescence assay confirmed that 2B protein could degrade RIG-I. in a dose-dependent manner. In order to determine the binding domain between 2B protein and RIG-I, we divided 2B gene into three segments, named 2B (1-50), 2B (50-100), 2B (100-150), and cotransfected 293-T cells with RIG-I plasmid. It was found that 2B (100-150), that is, 2B carboxyl terminal, can degrade RIG-I.. Then the relationship between 2B and endogenous RIG-I was studied. The results showed that 2B protein could inhibit the transcription and expression of endogenous RIG-I in a dose-dependent manner. The results showed that 2B (1-50)-2B amino terminal could inhibit the transcription of RIG-I mRNA. In order to understand the molecular mechanism of RIG-I degradation by direct interaction with RIG-I or by other means, indirect immunofluorescence, immunoprecipitation, proteasome and lysosome inhibition were also performed. The results showed that there was colocalization and interaction between 2B and RIG-I, and 2B degradation of RIG-I did not utilize the proteasome and lysosome pathway, but played a direct role in degradation by direct interaction with RIG-I. In conclusion, this study confirmed that porcine RIG-I had the function of resisting foot-and-mouth disease virus infection, and found that foot-and-mouth disease virus 2B protein could antagonize the innate immunity of porcine RIG-I against virus infection. The molecular mechanism of RIG-I degradation by 2B protein was preliminarily demonstrated. These results reveal the mechanism of immune escape of foot-and-mouth disease virus and provide a theoretical basis for further study on the molecular mechanism of host-pathogen interaction and the development of antiviral drugs and new vaccines.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
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