VISA相互作用蛋白的筛选与鉴定
发布时间：2018-11-09 21:33
【摘要】：病原微生物感染对人类健康构成巨大威胁,先天免疫系统是生物体在长期进化过程中建立起来的天然保护系统,是机体对抗外界病原体的第一道防线。VISA(MAVS,CARDIF,IPS-1)是连接胞浆dsRNA受体RIG-I与下游信号转导通路的一个接头蛋白,病毒入侵机体时,VISA在病毒激发的IFN-β信号通路起关键作用,VISA通过招募RIG-I样受体(包括RIG-I和MDA5)以及下游信号蛋白到线粒体上的VISA信号平台上,激活转录因子IRF3和NF-κB,从而诱导IFNs和其他炎症因子表达。研究结果表明VISA无论在TLR3非依赖的或者TLR3介导的抗病毒IFN信号途径中都具有关键作用,但目前对VISA信号转导的详细机制还不十分清楚。更多VISA相互作用蛋白的发现以及它们之间的作用机制的研究能完善我们对VISA参与的信号转导机制的认识。本研究利用酵母双杂交系统,用VISA蛋白的全长做诱饵(Bait)筛选293T细胞cDNA表达文库,并结合免疫共沉淀实验,双荧光素酶报告基因系统验证筛选到的阳性克隆和VISA作用的真实性。本研究成功筛选到Sec13L1候选基因并验证了其与VISA的全长蛋白相互作用,在293T细胞中过表达这个基因,我们发现Sec13L1过表达能促进VISA对IFNβ的诱导,并存在剂量效应。
[Abstract]:The infection of pathogenic microorganisms poses a great threat to human health. The innate immune system is a natural protective system established by organisms in the long evolution process and the first line of defense against external pathogens,. VISA (MAVS,CARDIF,. IPS-1) is a junction protein linking cytoplasmic dsRNA receptor RIG-I and downstream signal transduction pathway. VISA plays a key role in the IFN- 尾 signaling pathway stimulated by virus when the virus invades the organism. VISA induces the expression of IFNs and other inflammatory factors by recruiting RIG-I like receptors (including RIG-I and MDA5) and downstream signal proteins to the VISA signal platform on mitochondria to activate the transcription factors IRF3 and NF- 魏 B. The results show that VISA plays a key role in TLR3 independent or TLR3 mediated antiviral IFN signaling pathway, but the detailed mechanism of VISA signal transduction is not well understood. The discovery of more VISA interacting proteins and the study of their interaction mechanism can improve our understanding of the signal transduction mechanism in which VISA is involved. In this study, yeast two-hybrid system was used to screen 293T cell cDNA expression library using full-length (Bait) of VISA protein as bait, and combined with co-immunoprecipitation test. Double luciferase reporter gene system was used to verify the authenticity of the screened positive clones and the role of VISA. In this study, the candidate gene of Sec13L1 was successfully screened and its interaction with the full-length protein of VISA was verified. The gene was overexpressed in 293T cells. We found that overexpression of Sec13L1 promoted the induction of IFN 尾 by VISA, and there was a dose effect.
【学位授予单位】：江西师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392
本文编号：2321614
[Abstract]:The infection of pathogenic microorganisms poses a great threat to human health. The innate immune system is a natural protective system established by organisms in the long evolution process and the first line of defense against external pathogens,. VISA (MAVS,CARDIF,. IPS-1) is a junction protein linking cytoplasmic dsRNA receptor RIG-I and downstream signal transduction pathway. VISA plays a key role in the IFN- 尾 signaling pathway stimulated by virus when the virus invades the organism. VISA induces the expression of IFNs and other inflammatory factors by recruiting RIG-I like receptors (including RIG-I and MDA5) and downstream signal proteins to the VISA signal platform on mitochondria to activate the transcription factors IRF3 and NF- 魏 B. The results show that VISA plays a key role in TLR3 independent or TLR3 mediated antiviral IFN signaling pathway, but the detailed mechanism of VISA signal transduction is not well understood. The discovery of more VISA interacting proteins and the study of their interaction mechanism can improve our understanding of the signal transduction mechanism in which VISA is involved. In this study, yeast two-hybrid system was used to screen 293T cell cDNA expression library using full-length (Bait) of VISA protein as bait, and combined with co-immunoprecipitation test. Double luciferase reporter gene system was used to verify the authenticity of the screened positive clones and the role of VISA. In this study, the candidate gene of Sec13L1 was successfully screened and its interaction with the full-length protein of VISA was verified. The gene was overexpressed in 293T cells. We found that overexpression of Sec13L1 promoted the induction of IFN 尾 by VISA, and there was a dose effect.
【学位授予单位】：江西师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392
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