鮸鱼TLR受体新成员对革兰氏阴性菌LPS的识别及分子组成结构

Lipopolysaccharide (LPS) is one of the characteristic pathogen-associated molecular patterns (PAMPs) of Gram-negative bacteria, and the receptor for LPS is toll-like receptor 4 (TLR4) in mammals. The great threat for the aquaculture is the Gram-negative bacteria precisely. But most fishes lack TLR4 and the costimulatory molecules, and another receptor for LPS-receptor for advanced glycation end products (RAGE)-is also absent, leaving the question that which molecular is the receptor for LPS in fish. We speculate that fish TLR2 may be the receptor for the recognition of LPS and we firstly found a new member of TLR2 in miiuy croaker (Miichthys miiuy) genome. The first step of this project is to study the cellular localization of TLR2, and then through gene knockout and overexpression of TLR2 to test whether TLR2 recognize LPS. According to evolutionary theory to presume the molecular structure of TLR2 ligands, and co-immunoprecipitation and functional verification will be analysed to identify the type and molecular structure of ligands for TLR2. The results of this project will not only clarify functional molecular structure of fish TLR2 ligands but also determine whether fish TLR2 recognize LPS. And the findings will contribute to the immune prevention, and genetic breeding as well as the evolution of the immunity.

脂多糖LPS是革兰氏阴性菌的特征PAMP，哺乳动物中LPS的识别受体是TLR4分子。水产养殖中面临最多的致病病原就是革兰氏阴性菌，但鱼类中缺失TLR4和辅助TLR4识别LPS的共刺激分子，哺乳动物中也可以识别LPS的RAGE分子在鱼类中被证明也不存在，那么在鱼类中哪个受体分子可以识别LPS？分析推测在鱼类中TLR2可能是LPS的识别受体，申请人在鮸鱼全基因组中首次发现了一个TLR2受体新成员。本项目首先开展TLR2的细胞定位研究，然后通过基因敲降和过表达对TLR2是否是LPS的受体进行鉴定；根据免疫进化理论推测TLR2识别配体的分子结构类型，利用免疫共沉淀和功能验证彻底明确TLR2识别配体的分子组成结构及识别LPS的结构类型。本项目的研究结果不仅可以阐明鱼类TLR2发挥配体识别功能的分子结构以及判断是否作为LPS的识别受体，而且研究结果将有助于鱼类免疫防控和分子育种以及免疫进化的相关研究。

鱼类养殖中主要的致病病原菌是革兰氏阴性菌，革兰氏阴性菌能引起多种严重疾病，在哺乳动物中通过TLR4识别革兰氏阴性菌的LPS成分，但大部分鱼类中缺失TLR4，因此鉴定出鱼类免疫系统中能够识别革兰氏阴性菌组分的受体具有重要意义。申请人围绕鱼类革兰氏阴性菌识别受体的发现及其信号通路开展了系列研究，并取得系列研究成果。申请人发现了鱼类NOD1是革兰氏阴性菌LPS和iE-DAP的胞内识别受体，在鱼类致病革兰氏阴性菌感染下能够通过NOD1激活先天性免疫应答。进一步研究发现microRNA和lncRNA参与调控NOD1识别LPS后激活的信号通路这一过程，通过抑制或者提高NOD1的表达在激活免疫应答的同时维持着机体的免疫平衡，研究结果阐明了LPS的受体并发现了几个重要的调控分子，这些研究结果对于制定鱼类的免疫防控策略奠定了重要的基础。