口蹄疫病毒结构蛋白VP0抑制Ⅰ型干扰素信号通路的研究
发布时间：2019-01-18 16:03
【摘要】：目的:口蹄疫病毒在感染宿主细胞过程中,能诱导天然免疫应答产生。有文献报道口蹄疫病毒的非结构蛋白(L和3C)对天然免疫系统激活有直接阻断作用,但对结构蛋白VP0在抑制天然免疫反应中的作用机制尚不明确。本实验拟研究口蹄疫病毒(FMDV)结构蛋白VP0对Ⅰ型干扰素信号通路的影响。方法:通过反转录PCR构建VP0真核表达载体,利用western blot验证VP0蛋白转染HEK-293T细胞后的表达情况;Real-time PCR检测VP0蛋白与PVR蛋白分别对FMDV在BHK细胞上复制的影响,检测VP0蛋白与PVR蛋白分别对SeV诱导的干扰素信号通路分子RIG-I、IRF3、IFN-β及下游刺激基因ISG15、ISG20表达的影响;双荧光素酶报告基因检测系统检测VP0蛋白与PVR蛋白分别对SeV诱导的IFN-β和NF-κB启动子激活以及对RIG-I样受体(RIG-I-like receptors,RLRs)信号通路分子激活IFN启动子的影响;免疫共沉淀检测VP0蛋白、PVR蛋白分别与RLRs信号通路中关键分子的相互作用;间接免疫荧光检测VP0蛋白、PVR蛋白分别与关键分子IRF3在细胞中的定位。结果:成功构建了pCAGGs-VP0真核表达载体,可以在HEK-293T细胞中表达;FMDV感染4-6h后,VP0蛋白显著促进FMDV在BHK细胞上的复制(P0.01或P0.05),PVR蛋白可明显抑制FMDV的复制(P0.01或P0.05);VP0蛋白明显抑制干扰素下游刺激基因的表达(P0.01或P0.05),PVR蛋白对干扰素下游刺激基因的表达有促进作用(P0.01或P0.05)。在双荧光素酶报告基因检测实验中,VP0蛋白抑制SeV诱导IFN-β和NF-κB的活化有剂量依赖性(P0.01或P0.05),并对RIG-I、MDA5、VISA、TBK1和IRF3介导的IFN-β产生具有抑制作用(P0.05),但对IRF7没有明显影响;PVR蛋白可促进SeV介导的IFN-β和NF-κB的活化(P0.01或P0.05),并对RIG-I、MDA5、VISA、TBK1和IRF3介导的IFN-β产生有促进作用(P0.05),但对IRF7没有明显影响。免疫共沉淀显示VP0蛋白和PVR蛋白都可与IRF3发生相互作用;间接免疫荧光验证了VP0蛋白、PVR蛋白分别与IRF3在细胞中的共定位。结论:通过荧光定量PCR及双荧光素酶报告基因检测法证明了VP0蛋白可以显著抑制IFN-β和NF-κB的活化,而PVR蛋白可显著促进IFN-β和NF-κB的活化,从而推测FMDV结构蛋白VP0对Ⅰ型干扰素信号通路有抑制作用,PVR蛋白对Ⅰ型干扰素信号通路有促进作用。免疫共沉淀及激光共聚焦实验进一步证明了VP0蛋白和PVR蛋白可以分别通过与IRF3相互作用来抑制和促进Ⅰ型干扰素信号通路的激活,而VP0蛋白与PVR蛋白也可发生相互作用,据此可以认为这三种蛋白可能形成复合物,从而影响天然免疫系统。
[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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
本文编号：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.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
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