TGEV Nsp2 诱导宿主炎症反应的机制及其与病毒致病性的关系

Porcine transmissible gastroenteritis virus (TGEV) is an important etiological agent affecting swine industry worldwide. The research on TGEV pathogenesis is necessary to prevent and control this disease. It has been reported that TGEV infection induces acute inflammatory response in intestine, which is considered to be a central event in the pathogenesis of TGEV infection. NF-κB signal pathway plays a critical role in the inflammation that induces by virus infection. In a preliminary work of current study, we found that TGEV infection could activate NF-κB signal pathway and Nsp2 plays a key role. In this study, the mutants of Nsp2 will be constructed to further identify the key amino acids that associated with inflammation. Then, the host cellular proteins that interact with the Nsp2 and its mutant during TGEV infecting will be identified through the immunoprecipitation (IP) technique coupled with the LC-MS/MS, and the bioinformatics technology and the molecular biological technique will be used to clarify the molecular mechanisms of the inflammatory response that induced by Nsp2 expression. To test whether the amino acids of Nsp2 associated with inflammation promote TGEV pathogenesis, a recombinant TGEV cDNA clone harboring mutations in this motif was engineered and characterized. The results of this research will provide a novel way for researching the new therapeutic targets and designing the new generation vaccines to control TGEV.

猪传染性胃肠炎病毒（TGEV）所致仔猪病毒性腹泻给我国及世界养猪业造成了巨大的经济损失。TGEV感染突出的病理变化为肠道的卡他性炎症，由此可见，TGEV所致机体炎症反应的能力可能与其致病性密切相关。NF-κB信号通路在机体炎症反应发生过程中发挥主要作用，在本研究的前期工作中发现，TGEV感染可激活NF-κB信号通路，Nsp2对该通路具有显著的激活作用。据此，本研究将进一步确定Nsp2激活NF-κB信号通路介导炎症反应的关键位点，通过免疫沉淀结合串联质谱的方法筛选与Nsp2及其关键位点突变体相互作用的差异宿主细胞蛋白，并利用反向遗传学技术分析该位点与病毒致病性的关系，力图从病毒与宿主相互作用的角度揭示Nsp2诱导炎症反应的分子机理，确定TGEV Nsp2在病毒致病过程中所发挥的作用。本研究将为深入了解TGEV的致病机制以及TGEV 感染新型防控产品的开发提供理论基础和科学依据。

猪传染性胃肠炎病毒（TGEV）所致仔猪病毒性腹泻给我国及世界养猪业造成了巨大的经济损失。TGEV感染突出的病理变化为肠道的卡他性炎症，由此可见，TGEV所致机体炎症反应的能力可能与其致病机制密切相关。NF-κB信号通路在机体炎症反应发生过程中发挥主要作用，在本研究的前期工作中发现，TGEV感染可激活NF-κB信号通路，Nsp2对该通路具有显著的激活作用。据此，本项目进一步探索了TGEV Nsp2诱导宿主炎症反应的分子机制。本研究的结果表明，TGEV Nsp2 的1-120位氨基酸是激活NF-κB信号通路的关键序列。TGEV感染和Nsp2表达均可促进IκBα的降解及p65的磷酸化、核转移，由此可见，TGEV感染和Nsp2表达可通过经典途径激活NF-κB信号通路，从而诱导炎症反应发生。免疫沉淀结合串联质谱试验共鉴定出了84个与TGEV Nsp2互作的宿主细胞蛋白，生物信息学分析的结果表明，与TGEV Nsp2互作的宿主细胞蛋白主要参与细胞代谢过程、应激过程及对生物学过程的调控等。同时通过Co-IP和共定位试验进一步验证了TGEV Nsp2与宿主细胞蛋白ATP5B、PSMD11、Citrin和HSPA2之间的相互作用，证明了本研究筛选出的与TGEV Nsp2相互作用的蛋白具有较高的可信度。本研究结果将为揭示Nsp2在病毒致病过程中所发挥的作用提供重要线索和科学依据，为进一步探索TGEV的致病机制指出了新的方向。