不同品种山羊Toll样受体多态性与支原体肺炎易感相关性研究

Toll-like receptors are widely expressed in the mammalian cell surface as transmembrane signal transduction protein and are the Important molecules of the host immune system and the main receptors of natural immune pattern recognition. The preliminary research of our project group found that different goat breeds had different disease susceptibility(such as Goatpox and Mycoplasma pneumonia).The phenomenon remained us that may be connected with the innate immune response of goat breeds. Based on the above, the relationship between Toll like receptor polymorphism and the susceptibility of Mycoplasma in different breeds was presented. Guizhou local goat breeds and the introduced breeds are selected as the research objects, Mycoplasma pneumonia infected by Mycoplasma mycoides subspecies (Mmc) are as the epidemic model. The methods of molecular cloning and sequencing, fluorescence quantitative PCR, histopathology science, molecular immunology are used to detect the polymorphism of TLRs gene and their expression, different susceptibility for Mmc and the changes of toll-like receptors expression and signaling pathway key adaptor before and after infection. The results will clarify the mechanism of different goat breeds with different disease susceptible from the pathway of toll like receptor polymorphism and the relationship of Toll-like receptor regulating host immune and disease development.The research will lay the theoretical foundation for the analytical pathogenic mechanism of goats Mycoplasma pneumonia and a new way for disease prevention.

研究证实Toll样受体是广泛表达于哺乳动物细胞表面的一种跨膜信号转导蛋白，是宿主免疫的重要分子和天然免疫模式识别的主要受体。项目组前期研究发现不同品种山羊对疫病（如山羊痘和支原体肺炎）的易感性存在明显差异，分析认为与不同品种山羊天然免疫存在某种联系。据此提出不同品种山羊Toll样受体多态性与支原体易感相关性研究。选择贵州省地方品种山羊和引进山羊为研究对象，以丝状支原体山羊亚种（Mmc）支原体肺炎为疫病模型，采用分子克隆与测序、荧光定量PCR、组织病理学、分子免疫学等方法，检测不同品种山羊TLRs基因、表达多态性，Mmc易感差异性和感染前后Toll样受体表达、信号通路关键接头分子表达等调控变化。从Toll样受体多态性角度阐明不同品种山羊疫病易感机制，揭示Toll样受体调控宿主免疫与疫病发生相关关系，为解析山羊支原体肺炎致病机理奠定理论基础，为山羊抗病育种和疫病防控提供新思路。

Toll 样受体(Toll-like receptors, TLRs) 是动物进化中比较保守的一个受体家族，能特异地识别病原相关的PAMPs，不仅在激活天然免疫中发挥重要的作用，而且还调节获得性免疫，是连接天然免疫和获得性免疫的桥梁，从而有效地监测病原微生物的入侵以及诱导机体免疫应答反应。TLRs通过MyD88依赖途径和MyD88非依赖信号途径参与炎症反应。MyD88分子是大多数TLRs信号转导中的接头分子，可激活NF-κB和AP-1，控制炎症因子的分泌。项目组前期研究发现不同品种山羊对疫病（如山羊痘和支原体肺炎）的易感性存在明显差异，分析认为与不同品种山羊天然免疫存在某种联系。据此提出不同品种山羊Toll样受体多态性与支原体易感相关性研究。本研究发现，贵州黑山羊、贵州白山羊、黔北麻羊、波尔山羊和湖羊确实存在TLRs基因多态性、转录水平多态性，且某些多态性位点影响到TLRs二级结构、三级结构和功能结构域。动物人工感染试验显示不同品种羊对Mo感染存在明显不同，呈现为临床表现、死亡率、组织病变程度、组织病原核酸载量、炎症程度等差异，体现为炎症细胞数量、炎症分子表达量的差异，反映了不同品种羊感染Mo后的炎症反应程度、免疫反应程度、炎症进展速度及转归方式。通过对感染群体TLRs分子及其通路因子的转录水平分析认为，TLRs参与了Mo所致羊支原体肺炎的过程，通过多态性差异影响其MyD88通路、TRIF通路，调控不同品种羊对Mo易感性和炎症程度。