副猪嗜血杆菌OmpP2蛋白及其Loop诱导猪肺泡巨噬细胞免疫反应的作用和分子机制

Haemophilus parasuis (HPS) is an important respiratory-tract pathogen in swine. The outer membrane protein P2 (OmpP2) of HPS plays an important role in induction of pro-inflammatory reaction and immune protection, however, the molecular mechanisms were still unknown. There are predicted eight Loops existed in OmpP2 of virulence HPS strain. Due to the Loops involving in the immune reaction in other pathogenic bacteria, we speculate that the Loops of HPS OmpP2 also play a role in regulation of the immune responses. In this study, the HPS virulence SC096 strain OmpP2 and its Loops were used to stimulate the porcine alverolar macrophages to study the cytokines expression by Real-time PCR, Western blot, Dual-luciferase reporter system, RNA interference and other techniques. The study will confirm the functions of OmpP2 and Loops in proinflammatory reaction and immune protection. We will analyze the key molecular expressions in TLR/NF-κB and MAPK signal pathways to explore the OmpP2 and Loops to induce the immune responses mechanisms. Hence, the study will be able to explain the function and mechanisms of regulation the immune responses of the OmpP2 Loops, which play a significant role to further study the basic biology, pathogenic mechanism, prevention and control of HPS.

副猪嗜血杆菌（HPS）是危害全球养猪业一种重要的细菌性病原。HPS外膜蛋白P2（OmpP2）在诱导炎症反应和免疫保护中发挥重要作用，但分子机制不清楚。HPS强毒株OmpP2预测由8个表面环状结构（Loop）组成。鉴于其它致病菌Loop参与了免疫反应的作用，我们推测HPS OmpP2的Loop在免疫反应中发挥作用。本项目拟以HPS强毒株SC096株OmpP2和其Loop肽段为研究对象，刺激猪肺泡巨噬细胞，利用荧光定量PCR、Western blot、双荧光素酶报告系统和RNA干涉等技术，研究OmpP2和Loop肽段诱导细胞因子的表达，阐明在炎症反应和免疫保护中的作用；分析TLR/NF-κB和MAPK通路中关键分子的表达，探索诱导免疫反应的机制。本项目阐明HPS OmpP2和其Loop诱导免疫反应的作用和机制，对HPS基础生物学、致病机制和免疫防控的研究具有重要意义。

副猪嗜血杆菌（HPS）是危害全球养猪业一种重要的细菌性病原。HPS外膜蛋白P2（OmpP2）在诱导炎症反应和免疫保护中发挥重要作用，但分子机制不清楚。HPS强毒株OmpP2预测由8个表面环状结构（Loop）组成。鉴于其它致病菌Loop参与了免疫反应的作用，我们推测HPS OmpP2的Loop在免疫反应中发挥作用。本项目以HPS强毒株SC096株OmpP2和其Loop肽段为研究对象，刺激猪肺泡巨噬细胞，利用荧光定量PCR、Western blot、双荧光素酶报告系统和RNA干涉等技术，首次证实了loops L7和L8对诱导PAMs中细胞因子IL-1α，IL-1β，IL-6，IL-8，IL-17，IL-23，CCL-4和CCL-5的表达具有显著作用。发现了loops L7和L8作为OmpP2蛋白最活跃、最保守肽段，可能是开发潜在疫苗和抗HPS感染的抗菌药物的良好靶标。首次证实了loops L7和L8在参与OmpP2诱导炎症反应过程中，通过TLR1、TLR2、TLR4和TLR6途径激活NF-κB和MAPK信号通路。发现了loop L7通过调节p38、JNK、p65蛋白激活MAPK和NF-κB信号通路，参与OmpP2诱导细胞因子IL-1α，IL-8，IL-17，CCL-4 mRNA的表达; Loop L8通过调节p38蛋白激活MAPK信号通路，参与OmpP2诱导炎症反应，说明了loops L7和L8是OmpP2在诱导炎症反应过程中的分子位点，为抗HPS感染的抗菌药物的研制提供理论依据。因此，本研究首次从HPS OmpP2结构特征的角度分析loop肽段诱导宿主细胞炎症反应的作用和机制，确定HPS OmpP2在诱导宿主细胞炎症反应的分子位点，对HPS基础生物学、致病机制和免疫防控的研究具有重要意义。