猪细小病毒NS1通过Toll样受体调控细胞炎性应答的分子机制研究

Porcine parvovirus (PPV) is one of the major causes of reproductive failure disease in swine. Many mutants were emerging in recent years and PPV had highly co-infecting rate with other pathogens, which caused significant economic damage on the pig production. Our previous research showed that PPV and its nonstructural protein NS1 could active the nuclear factor kappa B (NF-κB) signal pathway by toll-like receptor 2,7 and 9 (TLR2,7,9), and regulated the host inflammatory response.These suggest that PPV NS1 is a pathogen associated molecular pattern during the PPV infection.However,the exact mechanism and precise process of the NS1 binding with TLRs,the molecular details of NS1 regulation on inflammatory response are not clear.Therefore,this project will focus on the recognition process of TLRs on the PPV NS1.Firstly,this project find the TLR types that combine with PPV NS1 protein and the receptors when NS1 as a ligand.Next,it analyzed the functional domain of TLRs with the truncations and mutant NS1, to reveal the molecular mechanism of the regulation of inflammatory response of PPV NS1 as a new PAMP.These investigations will provide scientific basis for the explanation of the pathogenic mechanism of PPV and also be beneficial for development of anti-PPV drug.

猪细小病毒（PPV）是引起母猪繁殖障碍为主要特征的重要病原体之一，近年来变异迅速，常与其它病原体混合感染，给养猪业造成了严重危害。项目组前期证实PPV及NS1均能通过Toll样受体（TLR） 2、7和9等TLRs激活NF-κB信号通路，进而调控宿主炎性反应，提示PPV NS1为新的病原相关分子模式（PAMP）。但NS1与TLRs结合过程、NS1与TLRs结合的结构基础以及NS1调控炎性反应的分子细节尚不清楚。据此，本项目以TLRs对PPV NS1识别过程为主题，首先明确能与PPV NS1结合的TLRs类型以及NS1作为配体的类型，其次解析PPV NS1截短体和突变体与TLRs功能域结合过程，进而探明PPV NS1作为新的PAMP及调控炎性应答的分子机制。为阐明PPV的致病机制提供理论依据，为进一步研发抗PPV病毒药物奠定理论基础。

猪细小病毒（porcine parvovirus, PPV）主要引起母猪的繁殖障碍，初产母猪产生死胎、弱胎和木乃伊胎，降低母猪的经济性能。目前对PPV的基因序列、结构及主要蛋白的功能已有较深的了解，但有关PPV感染引起猪的免疫机理及发病机制尚不清楚，严重制约新型疫苗和有效抗病毒药物的研发。因此，本研究对PPV感染后，宿主细胞的模式识别受体（pattern recognition receptors, PRRs）识别PPV病原相关分子模式（pathogen-associated molecular pattern, PAMP）、激活信号转导的过程进行研究。项目组前期证实PPV及NS1均能通过TLR2/TLR7/TLR9激活NF-κB信号通路，进而调控宿主炎性反应，提示PPV NS1为新的PAMP。据此，本项目以TLRs对PPV NS1识别过程为主题，明确能与PPV NS1结合的TLRs类型以及NS1作为配体的类型，其次解析PPV NS1截短体和突变体与TLRs功能域结合过程，进而探明PPV NS1作为新的PAMP及调控炎性应答的分子机制。用pCAGGS-HA-NS1重组质粒转染细胞，qPCR检测TLR2/TLR7/TLR9 mRNA的表达，确定TLR2是PPV NS1识别的主要TLRs受体。接着对PPV NS1和TLR2的三维立体结构进行模拟、分析，NS1与TLR2的200-300位氨基酸形成的结构域之间可能存在相互作用，设计PPV NS1和TLR2的截短体和突变体进行进一步验证。PPV NS1转染细胞后，wb检测发现IκBα发生降解，p65发生磷酸化，且TLR2参与PPV NS1介导的IκBα降解和p65磷酸化。本研究为阐明PPV的致病机制提供理论依据，同时也为抗PPV新型药物和疫苗的设计、临床防控提供理论依据，具有一定的应用前景。