口蹄疫病毒2B蛋白拮抗RIG-I抗病毒作用研究
發(fā)布時間：2018-10-14 18:13
【摘要】：口蹄疫病毒所引起的口蹄疫是偶蹄動物的烈性傳染病,世界動物衛(wèi)生組織(OIE)一直將其列為法定必報病,在我國被農(nóng)業(yè)部列為一類傳染病之首,口蹄疫的暴發(fā)往往會導(dǎo)致嚴(yán)重的經(jīng)濟損失。RIG-I樣受體是天然免疫系統(tǒng)中一類重要的模式識別受體,能夠識別細(xì)胞質(zhì)中的病毒RNA。RLR信號通路既受到宿主的嚴(yán)格調(diào)控,也能夠作為病毒逃逸宿主抗病毒反應(yīng)的靶點。目前,很多RNA病毒拮抗RIG-I抗病毒天然免疫的分子機制已經(jīng)研究清楚,而口蹄疫病毒對RIG-I抗病毒作用的拮抗機制研究尚不明朗。本研究以口蹄疫病毒拮抗RIG-I抗病毒天然免疫為切入點,構(gòu)建了豬RIG-I以及A型口蹄疫病毒蛋白(VP1、VP2、VP3、VP4、2B、3A、3B、3C、3D)的真核表達(dá)質(zhì)粒,并通過western blotting和間接免疫熒光實驗對構(gòu)建的真核表達(dá)質(zhì)粒進行了表達(dá)驗證,證明其成功表達(dá),同時證明豬RIG-I蛋白定位于細(xì)胞的胞漿中。感染實驗發(fā)現(xiàn)口蹄疫病毒能夠誘導(dǎo)細(xì)胞內(nèi)RIG-I的轉(zhuǎn)錄上調(diào),表明兩者間存在著重要聯(lián)系。過表達(dá)實驗證實RIG-I具有抑制FMDV復(fù)制的作用,而下調(diào)表達(dá)RIG-I可以促進FMDV的復(fù)制,表明RIG-I在機體抗口蹄疫病毒感染過程中發(fā)揮著重要的作用。同時,在口蹄疫病毒感染過程中,還發(fā)現(xiàn)RIG-I在蛋白水平上隨感染時間的延長會被逐漸降解。隨后將構(gòu)建好的口蹄疫病毒各個病毒蛋白質(zhì)粒分別與RIG-I質(zhì)粒共轉(zhuǎn)染293-T細(xì)胞,發(fā)現(xiàn)口蹄疫病毒2B蛋白可以降解RIG-I,而且通過western blotting和間接免疫熒光實驗證實2B蛋白可以劑量依賴性的降解RIG-I。為了確定2B蛋白與RIG-I互作的結(jié)合域,本研究將2B基因分為三段分別構(gòu)建了突變體質(zhì)粒,命名為2B(1-50)、2B(50-100)、2B(100-150),接著將2B各個突變體質(zhì)粒分別與RIG-I質(zhì)粒共轉(zhuǎn)染293-T細(xì)胞,發(fā)現(xiàn)2B(100-150)即2B羧基端可以降解RIG-I。然后又對2B和內(nèi)源性RIG-I的關(guān)系進行了研究,結(jié)果發(fā)現(xiàn)2B蛋白可以劑量依賴性的抑制內(nèi)源性RIG-I的轉(zhuǎn)錄和表達(dá),進一步開展了2B及其突變體過表達(dá)實驗,結(jié)果證實2B(1-50)即2B氨基端可以抑制RIG-I mRNA的轉(zhuǎn)錄。為了探明2B是通過與RIG-I直接互作還是通過其他途徑來進行降解RIG-I的分子機制,筆者又做了間接免疫熒光、免疫共沉淀以及蛋白酶體和溶酶體抑制實驗,結(jié)果顯示2B和RIG-I存在共定位并發(fā)生互作,并且2B降解RIG-I不利用蛋白酶體和溶酶體途徑,而是通過與RIG-I直接互作發(fā)揮降解作用�？傊�,本研究證實了豬RIG-I具有抗口蹄疫病毒感染的功能,并且發(fā)現(xiàn)口蹄疫病毒2B蛋白具有拮抗豬RIG-I抗病毒天然免疫的作用,初步探明了2B蛋白降解RIG-I的分子機制。這些研究結(jié)果,揭示了口蹄疫病毒免疫逃逸的機制,為進一步研究宿主與病原間相互作用的分子機制以及開發(fā)抗病毒藥物和研制新型疫苗提供了理論依據(jù)。
[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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65
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