外膜囊泡递呈DNA在副猪嗜血杆菌诱导I型干扰素产生中的作用与分子机制研究

副猪嗜血杆菌(HPS)病是威胁我国养猪业发展的重要细菌性疾病，外膜囊泡(OMVs)作为HPS的新型独立分泌系统，与HPS和宿主相互作用及致病机制密切相关，但其分子机制尚不明确。本项目前期发现，OMVs富含HPS的DNA，参与HPS诱导巨噬细胞产生I型干扰素，且cGAS可能是OMVs诱导I型干扰素产生的关键节点。基于此，本项目拟先利用报告基因等方法比较HPS、OMVs、无核酸OMVs对I型干扰素产生的影响，明确OMVs携带DNA在HPS诱导巨噬细胞产生I型干扰素中的作用；通过免疫印迹、凝胶阻滞、激光共聚焦和慢病毒介导的CRISPR-Cas9等方法，进一步阐明OMVs递呈DNA激活cGAS-STING信号通路诱导I型干扰素产生的关键分子机制；最后通过基因缺失小鼠解析OMVs携带DNA诱导I型干扰素对HPS致病力的影响，从一个新的角度揭示HPS的致病机制，为HPS病的防控提供理论依据。

Haemophilus parasuis (HPS) disease is one of important bacterial diseases which threaten the development of pig industry in China. Outer Membrane Vesicle (OMV) is a new independent secretion system of HPS, which plays an important role in the interaction between bacteria and host cells and associates with bacterial pathogenicity, but the molecular mechanism of this process is still unclear. The previous study of this project showed that OMVs of HPS carried a large amount of DNA, and involved in the production of type I interferon. Moreover, cGAS might be the key node in OMVs-induced type I interferon production. Therefore, this project intends to use Luciferase reporter gene, Q-PCR and ELISA to determine whether the DNA carried by OMVs is the key factor for HPS inducing macrophages to produce type I interferon. Subsequently, we will continue to study the key molecular mechanism of type I interferon production by the OMVs-mediated DNA-activated cGAS-STING signaling pathway. Western blot, EMSA, confocal and lentiviral-mediated CRISPR/Cas9 genome editing technique will be used. Finally, the influence of OMVs carries DNA to induce type I interferon production on the pathogenicity of HPS was studied by gene knockout mice. This project will reveal the pathogenicity mechanism of HPS from a new perspective and provide theoretical basis for the prevention and control of HPS disease.