课题组前期研究发现在免疫分子RIG-I和IRF3天然缺失的鸡细胞中，MDA5与STING互作构成了一条在哺乳动物中并不存在的MDA5-STING-IFNβ天然免疫信号通路，但对该通路具体的信号传导和抗病毒机制尚不清楚。本项目将聚焦此通路，以鸡STING为核心，通过免疫共沉淀、质谱分析、RNA干扰和蛋白核酸互作等技术，分析STING与MDA5等信号分子互作的分子基础；阐明其亚细胞定位依赖的功能域及其生物学意义；解析STING介导信号传导依赖的泛素化、磷酸化等修饰机制；揭示鸡STING激活IFNβ依赖的转录因子。应用CRISPR-Cas9技术构建STING基因敲除鸡细胞系，进而基于H9N2禽流感病毒感染后的复制水平和免疫分子表达差异，揭示鸡STING介导的抗病毒途径。通过研究，初步阐明鸡I型干扰素信号通路中STING介导信号传导和抗病毒的分子机制，为利用鸡天然免疫能力抗感染奠定理论和应用基础。

It was previously found that MDA5 (mammalian melanoma differentiation associated gene 5, MDA5) could interact with STING (stimulator of interferon genes, STING) to reconstitute a new MDA5-STING-IFNβ signal pathway in RIG-I(retinoic acid inducible gene I, RIG-I) and IRF3(IFN regulatory factor 3, IRF3) absent chicken cells,but not in mammalian cells. However, the mechanisms of signal transduction and antiviral effects of this pathway remains unclear. This proposal will focus on the analysis of typeⅠinterferon pathway mediated by chicken MDA5（chMDA5）and chicken STING (chSTING), and will highlight the function of chSTING. So, the molecular and structural basis of interaction between chMDA5 and chSTING will be solved, and the mechanism of posttranslation modification of chSTING will be analyzed. The IFN regulatory factor required for activation of beta IFN by chSTING will be investigated by using immunoprecipitation and mass spectrometry combined assay, RNA interference technique and protein-nucleic acid interaction technique,such as electrophoretic mobility shift assay (EMSA). To determine the roles of chSTING in inhibition of H9N2 avian influenza virus(AIV) replication, the virus titers will be measured in CRISPR-Cas9 mediated chSTING gene knocked out and mock chicken cells. Combined with gene chip and qRT-PCR technology, the antiviral immune genes related to chSTING in the process of H9N2 AIV infection will also be characterized. These findings will allow the chicken specific MDA5-STING-IFNβsignaling pathway to be clearly illustrated and the mechanism of chSTING in antivirus to be determined,which is crucial for the prevention and control of AIV and other viral infections.