三疣梭子蟹补体凝集素激活途径及其介导的母源免疫机制研究

Complement lectin pathway is the important component of innate immunity. The study on invertebrate complement system started relatively late and the information about complements in crustaceans remains deficient, especially in brachyuran crabs. In our previous study, eight molecules related to complement lectin pathway have been obtained from the swimming crab Portunus trituberculatus. In this project, the complement lectin pathway will be further investigated. The function of their recombinant proteins will be analyzed by the biological activies, such as hemagglutination, microbial aggregation, antimicrobial activity, protease activity and Vibrio clearance. Their expression pattern during different tissues and development stages of P. trituberculatus will be elucidated by molecular biotechnologies including quantitative Real-time PCR, Western blot and immunohistochemistry. The mediated cell phagocytosis and activation mechanism of the complement lectin pathway will be investigated by Pull down and co-immunoprecipitation. The complement binding proteins and their binding domain will be identified. After interfering the key genes, the change on the hemocytes phagocytosis rate, the total hemolymph immune response and the expression levels of regulatory genes will be checked. The components and their function of the maternal complement lectin pathway will be examined by the experiments of blocking antibody and antibacterial activity. The obtained results will clarify the molecular components and function of the complement lectin pathway in P. trituberculatus. It will provide valuable reference for understanding maternal immunity of crab and its trans-generational effect. This study will provide more clues for the evolution of complement system and will be also useful in giving new insights into crustacean immune defense mechanism.

补体凝集素途径是固有免疫的重要组成部分，然而无脊椎动物补体研究起步较晚，迄今对甲壳动物特别是蟹类补体的研究尚属空白。本项目在获得三疣梭子蟹8种凝集素途径分子基础上，验证其重组蛋白的凝血、凝菌、抑菌、蛋白酶、弧菌清除等生物学活性；应用RT-PCR、原位杂交、免疫组化等技术，明确这些基因在三疣梭子蟹各组织和胚胎发育时期的时空表达规律；应用Pull down、Co-IP等技术，鉴定凝集素途径的互作分子、明确其互作结构域，同时应用RNAi阻断关键补体成分，检测血细胞吞噬率、免疫酶活及调控基因表达的差异，解析其介导的细胞吞噬和激活调控通路；通过抗体阻断、抗菌活性检测等实验，分析母源凝集素途径的构成及其对后代的免疫保护机制。本项目将阐明三疣梭子蟹补体凝集素激活途径的分子组成及功能，为理解蟹类母源免疫及其传代效应奠定基础，对明晰补体系统进化脉络、揭示甲壳动物免疫防御机制具有重要意义。

本项目按照计划完成了预定研究内容和各项考核指标，明确了三疣梭子蟹补体凝集素途径10种分子的结构和主要的免疫功能，阐明了它们在三疣梭子蟹各组织和胚胎发育阶段的时空表达规律，重点探究了它们介导的细胞吞噬和激活调控通路，解析了三疣梭子蟹和中华绒螯蟹早期胚胎的免疫功能和母源补体对蟹类胚胎的免疫保护作用。共发表SCI论文7篇，授权发明专利1项，申请发明专利5项，培养研究生5人，参加国内外会议3次。. 系统研究了三疣梭子蟹10种补体凝集素途径分子包括C1q受体gC1qR（PtgC1qR）、α2-巨球蛋白（PtA2M-1和PtA2M-2）、含硫酯蛋白（PtTEP）甘露糖结合凝集素（PtMBL1和PtMBL2）、C型凝集素（PtClec1和PtClec2）和纤维蛋白原相关蛋白（Ptficolin和PtTachylectin）的序列结构和免疫功能，发现大多数补体样分子在卵巢和受精卵时期表达量较高，推测三疣梭子蟹的补体样分子可能参与母源性传递过程；重组表达的补体样分子具有广谱的抗菌、菌结合活性，能够在体外促进对弧菌的清除活性和在体内促进血细胞对弧菌的吞噬作用；补体样分子敲降后，吞噬相关基因、酚氧化酶原激活系统的相关基因、其它补体样分子、抗菌肽以及Toll通路和IMD通路关键基因显著变化，表明补体样分子可能通过调控吞噬、酚氧化酶原系统的激活、抗菌肽生成发挥免疫功能；三疣梭子蟹和中华绒螯蟹的受精卵胞浆蛋白提取物具有显著的抑菌、菌凝集和菌损伤活性，通过ELISA、金属离子螯合等实验明确了补体凝集素途径在胚胎的免疫保护中发挥重要作用。以上研究成果揭示了三疣梭子蟹补体凝集素途径的分子组成和调控机制，阐明了补体凝集素途径在母源免疫中的保护作用，有助于深入解析甲壳动物的免疫防御机制，亦为蟹类的健康养殖提供理论指导。