口蹄疫病毒結(jié)構(gòu)蛋白VP0抑制Ⅰ型干擾素信號通路的研究
發(fā)布時間：2019-01-18 16:03
【摘要】：目的:口蹄疫病毒在感染宿主細(xì)胞過程中,能誘導(dǎo)天然免疫應(yīng)答產(chǎn)生。有文獻(xiàn)報道口蹄疫病毒的非結(jié)構(gòu)蛋白(L和3C)對天然免疫系統(tǒng)激活有直接阻斷作用,但對結(jié)構(gòu)蛋白VP0在抑制天然免疫反應(yīng)中的作用機(jī)制尚不明確。本實驗擬研究口蹄疫病毒(FMDV)結(jié)構(gòu)蛋白VP0對Ⅰ型干擾素信號通路的影響。方法:通過反轉(zhuǎn)錄PCR構(gòu)建VP0真核表達(dá)載體,利用western blot驗證VP0蛋白轉(zhuǎn)染HEK-293T細(xì)胞后的表達(dá)情況;Real-time PCR檢測VP0蛋白與PVR蛋白分別對FMDV在BHK細(xì)胞上復(fù)制的影響,檢測VP0蛋白與PVR蛋白分別對SeV誘導(dǎo)的干擾素信號通路分子RIG-I、IRF3、IFN-β及下游刺激基因ISG15、ISG20表達(dá)的影響;雙熒光素酶報告基因檢測系統(tǒng)檢測VP0蛋白與PVR蛋白分別對SeV誘導(dǎo)的IFN-β和NF-κB啟動子激活以及對RIG-I樣受體(RIG-I-like receptors,RLRs)信號通路分子激活I(lǐng)FN啟動子的影響;免疫共沉淀檢測VP0蛋白、PVR蛋白分別與RLRs信號通路中關(guān)鍵分子的相互作用;間接免疫熒光檢測VP0蛋白、PVR蛋白分別與關(guān)鍵分子IRF3在細(xì)胞中的定位。結(jié)果:成功構(gòu)建了pCAGGs-VP0真核表達(dá)載體,可以在HEK-293T細(xì)胞中表達(dá);FMDV感染4-6h后,VP0蛋白顯著促進(jìn)FMDV在BHK細(xì)胞上的復(fù)制(P0.01或P0.05),PVR蛋白可明顯抑制FMDV的復(fù)制(P0.01或P0.05);VP0蛋白明顯抑制干擾素下游刺激基因的表達(dá)(P0.01或P0.05),PVR蛋白對干擾素下游刺激基因的表達(dá)有促進(jìn)作用(P0.01或P0.05)。在雙熒光素酶報告基因檢測實驗中,VP0蛋白抑制SeV誘導(dǎo)IFN-β和NF-κB的活化有劑量依賴性(P0.01或P0.05),并對RIG-I、MDA5、VISA、TBK1和IRF3介導(dǎo)的IFN-β產(chǎn)生具有抑制作用(P0.05),但對IRF7沒有明顯影響;PVR蛋白可促進(jìn)SeV介導(dǎo)的IFN-β和NF-κB的活化(P0.01或P0.05),并對RIG-I、MDA5、VISA、TBK1和IRF3介導(dǎo)的IFN-β產(chǎn)生有促進(jìn)作用(P0.05),但對IRF7沒有明顯影響。免疫共沉淀顯示VP0蛋白和PVR蛋白都可與IRF3發(fā)生相互作用;間接免疫熒光驗證了VP0蛋白、PVR蛋白分別與IRF3在細(xì)胞中的共定位。結(jié)論:通過熒光定量PCR及雙熒光素酶報告基因檢測法證明了VP0蛋白可以顯著抑制IFN-β和NF-κB的活化,而PVR蛋白可顯著促進(jìn)IFN-β和NF-κB的活化,從而推測FMDV結(jié)構(gòu)蛋白VP0對Ⅰ型干擾素信號通路有抑制作用,PVR蛋白對Ⅰ型干擾素信號通路有促進(jìn)作用。免疫共沉淀及激光共聚焦實驗進(jìn)一步證明了VP0蛋白和PVR蛋白可以分別通過與IRF3相互作用來抑制和促進(jìn)Ⅰ型干擾素信號通路的激活,而VP0蛋白與PVR蛋白也可發(fā)生相互作用,據(jù)此可以認(rèn)為這三種蛋白可能形成復(fù)合物,從而影響天然免疫系統(tǒng)。
[Abstract]:Objective: Foot-and-mouth disease virus (FMDV) can induce innate immune response in host cells. It has been reported that the non-structural proteins (L and 3C) of foot-and-mouth disease virus (FMDV) can directly block the activation of innate immune system, but the mechanism of the inhibitory effect of structural protein VP0 on innate immune response is unclear. The purpose of this study was to investigate the effect of foot-and-mouth disease virus (FMDV) (FMDV) structural protein VP0 on interferon type I signaling pathway. Methods: the eukaryotic expression vector of VP0 was constructed by reverse transcription PCR, and the expression of VP0 protein in HEK-293T cells was verified by western blot. Real-time PCR was used to detect the effects of VP0 protein and PVR protein on the replication of FMDV on BHK cells, and VP0 protein and PVR protein on SeV induced interferon signaling pathway RIG-I,IRF3,IFN- 尾 and downstream stimulating gene ISG15, respectively. The effect of ISG20 expression; The effects of VP0 protein and PVR protein on the activation of IFN- 尾 and NF- 魏 B promoter induced by SeV and IFN promoter activated by RIG-I like receptor (RIG-I-like receptors,RLRs) signaling pathway were detected by double luciferase reporter gene detection system. The interaction of VP0 protein and PVR protein with key molecules in RLRs signaling pathway was detected by immunoprecipitation, and the localization of VP0 protein and PVR protein with IRF3 was detected by indirect immunofluorescence. Results: pCAGGs-VP0 eukaryotic expression vector was successfully constructed and could be expressed in HEK-293T cells. After 4-6 hours of FMDV infection, VP0 protein significantly promoted the replication of FMDV on BHK cells (P0.01 or P0.05), PVR protein significantly inhibited FMDV replication (P0.01 or P0.05). VP0 protein significantly inhibited the expression of downstream stimulating gene of interferon (P0.01 or P05), PVR protein promoted the expression of downstream stimulating gene of interferon (P0.01 or P0.05). In the detection of double luciferase reporter gene, VP0 protein inhibited the activation of IFN- 尾 and NF- 魏 B induced by SeV in a dose-dependent manner (P0.01 or P0.05). The production of IFN- 尾 mediated by TBK1 and IRF3 was inhibited (P0.05), but it had no effect on IRF7. PVR protein could promote the activation of IFN- 尾 and NF- 魏 B mediated by SeV (P0.01 or P0.05) and IFN- 尾 production mediated by RIG-I,MDA5,VISA,TBK1 and IRF3 (P0.05), but had no effect on IRF7. Immunoprecipitation showed that VP0 protein and PVR protein could interact with IRF3, and indirect immunofluorescence confirmed the colocalization of VP0 protein, PVR protein and IRF3 in cells. Conclusion: fluorescence quantitative PCR and double luciferase reporter gene detection showed that VP0 protein could significantly inhibit the activation of IFN- 尾 and NF- 魏 B, while PVR protein could significantly promote the activation of IFN- 尾 and NF- 魏 B. It is inferred that FMDV structural protein VP0 can inhibit the type I interferon signaling pathway, and PVR protein can promote the type 1 interferon signaling pathway. The immunoprecipitation and laser confocal experiments further demonstrated that VP0 protein and PVR protein could inhibit and promote the activation of interferon I signaling pathway by interacting with IRF3, while VP0 protein and PVR protein could interact with each other. It can be concluded that these three proteins may form complex, thus affecting the innate immune system.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65
本文編號：2410877
[Abstract]:Objective: Foot-and-mouth disease virus (FMDV) can induce innate immune response in host cells. It has been reported that the non-structural proteins (L and 3C) of foot-and-mouth disease virus (FMDV) can directly block the activation of innate immune system, but the mechanism of the inhibitory effect of structural protein VP0 on innate immune response is unclear. The purpose of this study was to investigate the effect of foot-and-mouth disease virus (FMDV) (FMDV) structural protein VP0 on interferon type I signaling pathway. Methods: the eukaryotic expression vector of VP0 was constructed by reverse transcription PCR, and the expression of VP0 protein in HEK-293T cells was verified by western blot. Real-time PCR was used to detect the effects of VP0 protein and PVR protein on the replication of FMDV on BHK cells, and VP0 protein and PVR protein on SeV induced interferon signaling pathway RIG-I,IRF3,IFN- 尾 and downstream stimulating gene ISG15, respectively. The effect of ISG20 expression; The effects of VP0 protein and PVR protein on the activation of IFN- 尾 and NF- 魏 B promoter induced by SeV and IFN promoter activated by RIG-I like receptor (RIG-I-like receptors,RLRs) signaling pathway were detected by double luciferase reporter gene detection system. The interaction of VP0 protein and PVR protein with key molecules in RLRs signaling pathway was detected by immunoprecipitation, and the localization of VP0 protein and PVR protein with IRF3 was detected by indirect immunofluorescence. Results: pCAGGs-VP0 eukaryotic expression vector was successfully constructed and could be expressed in HEK-293T cells. After 4-6 hours of FMDV infection, VP0 protein significantly promoted the replication of FMDV on BHK cells (P0.01 or P0.05), PVR protein significantly inhibited FMDV replication (P0.01 or P0.05). VP0 protein significantly inhibited the expression of downstream stimulating gene of interferon (P0.01 or P05), PVR protein promoted the expression of downstream stimulating gene of interferon (P0.01 or P0.05). In the detection of double luciferase reporter gene, VP0 protein inhibited the activation of IFN- 尾 and NF- 魏 B induced by SeV in a dose-dependent manner (P0.01 or P0.05). The production of IFN- 尾 mediated by TBK1 and IRF3 was inhibited (P0.05), but it had no effect on IRF7. PVR protein could promote the activation of IFN- 尾 and NF- 魏 B mediated by SeV (P0.01 or P0.05) and IFN- 尾 production mediated by RIG-I,MDA5,VISA,TBK1 and IRF3 (P0.05), but had no effect on IRF7. Immunoprecipitation showed that VP0 protein and PVR protein could interact with IRF3, and indirect immunofluorescence confirmed the colocalization of VP0 protein, PVR protein and IRF3 in cells. Conclusion: fluorescence quantitative PCR and double luciferase reporter gene detection showed that VP0 protein could significantly inhibit the activation of IFN- 尾 and NF- 魏 B, while PVR protein could significantly promote the activation of IFN- 尾 and NF- 魏 B. It is inferred that FMDV structural protein VP0 can inhibit the type I interferon signaling pathway, and PVR protein can promote the type 1 interferon signaling pathway. The immunoprecipitation and laser confocal experiments further demonstrated that VP0 protein and PVR protein could inhibit and promote the activation of interferon I signaling pathway by interacting with IRF3, while VP0 protein and PVR protein could interact with each other. It can be concluded that these three proteins may form complex, thus affecting the innate immune system.
【學(xué)位授予單位】：蘭州大學(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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