七鳃鳗血清免疫防御病原体的途径及分子机制研究

七鳃鳗是现存最原始的脊椎动物之一，在长期水生生活中如何防御病原体的攻击尚未阐述。前期工作发现，七鳃鳗血清具有高效地杀伤病原体的活性，使病原体细胞破裂，内容物外泄，导致病原体死亡；通过病原体膜蛋白负染结合透射电镜观察到靶细胞膜上形成攻膜复合体结构，与补体C9攻膜复合体成孔形态上有较大差异，因此鉴定七鳃鳗血清攻膜蛋白（LSMAP）的结构，探究补体系统中重要免疫分子的协同作用及裂解病原体效应机制具有重要意义。本项目利用冷冻电镜观察攻膜复合体的分子结构，解析LSMAP杀伤病原体的作用特点；通过免疫共沉淀及非标记分子互作方法，研究参与攻膜复合体形成的相互作用分子；通过基因敲除及补体组分的替换、穿插技术，明确七鳃鳗免疫防御的分子机制。为阐释原始脊椎动物分子免疫学机制、水产动物病害免疫防御药物研发以及补体系统的分子起源与进化规律，提供科学依据和新的思路与方法。

Lamprey is the important animal model for studying the origin of the vertebrate immune system. It is still unknown that how lamprey stays alive and protects itself against pathogen attack. Our previous study revealed that the lamprey serum possess the cytocidal activity through the disruption of cell membrane and the leakage of cytoplasm, leading to the death of pathogen. The structure of the target cell membrane attacking complex was observed by negative staining of pathogenic membrane proteins combined with transmission electron microscopy, which is quite different from the pore-forming morphology of complement C9 attacking complex. Therefore, it is essential to identify the structure of lamprey serum membrane protein (LSMAP) and explore the synergistic effect of crucial immune molecules in the complement system and the mechanism of lytic pathogen effect. In this project, the molecular structure of the attack complex was observed by cryo-electron microscopy, and the characteristics of LSMAP killing pathogens were analyzed. The interaction molecules involved in the formation of membrane attack complex were examined by immunoprecipitation and non-labeled molecular interaction methods. And the molecular mechanism of immune defense of lamprey was clarified by gene knockout, complement replacement and interpolation techniques. In summary, our research provides a scientific basis and new ideas and methods in order to interpret the molecular immune mechanisms of primitive vertebrates, the development of immune defense drugs for aquatic animal diseases, and the molecular origin and evolution of the complement system.