中文摘要
猪瘟病毒感染能够导致猪免疫系统受到抑制，是严重危害全球养猪业的重要病原。最近研究发现，猪瘟病毒非结构蛋白Npro通过降解干扰素调节因子3（IRF3）来阻断I型干扰素（IFN）合成，但具体机制目前尚未阐明。本研究以此为切入点，利用GST Pull down，蛋白质质谱分析、哺乳细胞双杂交、免疫共沉淀、共聚焦显微镜等技术，筛选鉴定细胞中与猪瘟病毒Npro和/或IRF3相互作用的E3 ligases和靶位点；利用过量表达、SiRNA试验和荧光素酶报告系统，评价该E3 ligases在猪瘟病毒Npro降解IRF3中发挥的功能以及对IFN合成的影响。在此基础上，利用反向遗传学技术构建猪瘟病毒Npro突变株，分别在细胞和猪体内研究该突变株对I型IFN信号通路的影响。旨在阐明猪瘟病毒Npro蛋白抑制I型IFN产生的分子机制，为深入探讨猪瘟病毒分子免疫机制、致病机理和安全、高效新型疫苗研制奠定基础。
英文摘要
Classical swine fever virus(CSFV) is the significant etiological agent of Classical swine fever(CSF), a highly contagious disease in pig farm, which causes important economic losses worldwide. The infection of CSFV inhibit the host’s immune system. A recent study revealed that Npro, a nonstructure protein of CSFV, inhibits the production of type I interferon(IFNs) by targeting IFN regulatory factor 3(IRF3), but the mechanism is not clarified to date. In the study, we will screen and evaluate the E3 ligase and targets site in host cell, which can interact with Npro and/or IRF3 by GST pull down, preotein mass spectrography analysis, cytoTrap two-hybrid system, immunoprecipitation and confocal microscop. Meanwhile, overexpression, SiRNA and luciferase report system will be used to evaluate E3 ligase’s function on the degradation of IRF3 and on the synthesis of IFN. Furthermore, Npro- mutants will be constructed by reverse genetics technique, the mutants’s effects on the sigal pathway of type I IFN will be further studied on the level of in vivo and in vitro, respectively. Our aim is to illuminate the molecular mechanism of Npro on how to inhibit the production of type I IFN, and to further reveal CSFV’ molecular immunologic mechanism, pathogenesis mechanism and develop new generation of vaccine with more safety and higher performance.
结题摘要
猪瘟病毒感染能够导致猪免疫系统受到抑制，是严重危害全球养猪业的重要病原。最近研究发现，猪瘟病毒非结构蛋白Npro通过降解干扰素调节因子3（IRF3）来阻断I型干扰素（IFN）合成，但具体机制目前尚未阐明。本研究以此为切入点，利用GST Pull down，蛋白质质谱分析、哺乳细胞双杂交、免疫共沉淀、共聚焦显微镜等技术，筛选鉴定细胞中与猪瘟病毒Npro和/或IRF3相互作用的E3 ligases和靶位点；利用过量表达、SiRNA试验和荧光素酶报告系统，评价该E3 ligases在猪瘟病毒Npro降解IRF3中发挥的功能以及对IFN合成的影响。在此基础上，利用反向遗传学技术构建猪瘟病毒Npro突变株，分别在细胞和猪体内研究该突变株对I型IFN信号通路的影响。旨在阐明猪瘟病毒Npro蛋白抑制I型IFN产生的分子机制，为深入探讨猪瘟病毒分子免疫机制、致病机理和安全、高效新型疫苗研制奠定基础。