介导滑液支原体入侵的细胞表面蛋白的发现及机制研究

滑液支原体（MS）是禽类的一种重要致病性支原体，可引起鸡和火鸡的上呼吸道疾病、滑膜炎及气囊炎等，给世界养鸡业造成了巨大的经济损失。目前，MS感染的防控依旧是世界难题，阐明其感染机制和免疫机理则是解决这一难题的关键。为深入研究MS感染及免疫的分子机制，本研究拟利用Co-IP联合质谱分析筛选MS与细胞（DF-1）互作蛋白，继而以原核表达的MS蛋白为诱饵蛋白，经Pull-down联合质谱鉴定与诱饵蛋白互作的DF-1细胞膜蛋白，并在目的蛋白原核表达及其多抗制备的基础上，应用Western blot、粘附抑制试验和等温滴定量热法（ITC）验证目标蛋白间的互作，确定与MS互作的细胞表面蛋白，进而基于靶基因敲除细胞系的构建及MS内吞入侵抑制试验，对其介导MS入侵的机制进行解析。本项目将为深入研究MS感染及致病的分子机理和免疫机制奠定基础，也为MS感染防控用新产品、新方法和新技术的研发提供理论支持。

Mycoplasma synoviae(MS), one of major lethal avian pathogenic Mycoplasma, causes respiratory diseases, synovitis and airsacculitis in chickens and turkeys, and thus brings about substantial economic loss to the poultry industry throughout the world. At present, how to prevent and control MS infection is still a worldwide problem. Therefore, the illumination of the mechanism of infection and immunity to MS is the key to solving such problem. To further study the molecular mechanisms of MS infection and immunity, this study will screen the interaction proteins between MS and DF-1 cells by using co-immunoprecipitation (Co-IP) first and then by mass spectroscopy assay. Then the prokaryotic expressed MS membrane protein will be used as bait protein, and the membrane proteins of DF-1 cells that interact with bait protein will be screened and identified by utilizing Pull-down combined with mass spectroscopy assay. Subsequently, the target proteins from DF-1 cells will be expressed in prokaryotic expression system and the polyclonal antibody against recombinant proteins were prepared. Based on this, the interaction between two types of recombinant proteins will be further verified by Western blot, adherence inhibition assays and isothermal titration calorimetry (ITC). Finally, the mechanisms of those proteins mediated MS invading cells are finalized by using target gene knockout cells and MS endocytosis invasion inhibition test. The study will lay a foundation for further research on the pathogenesis and immune mechanism of MS, and provide theoretical support for the development of new products, new methods and new technologies to diagnose, prevent and control MS infection.