罗非鱼嗜中性粒细胞Siglecs与链球菌的相互作用及其免疫负调控机制研究

Streptococcal-infection has caused large scale motalilty of cultured tilapia in China since 2009, and seriously threaten the tilapia industry of China. In the previous study, our group had identified the pathogen and its serotype, as well as sequence type. The pathogen was Streptococcus agalactiae, which also designated group B streptococcus (GBS). It had the same serotype and sequence type with one of the common pathogen strain infected human beings, GBS ?a, ST-7. Molecular and immunological methods will be applied in this project to study the characteristics of Siglecs of tilapia. Siglecs cDNAs containing inhibitory motif ITIM will be isolated. Recombinant protein Siglecs-Fc will be prepared and used to assay the specific recognition and binding of Siglecs with sialic acid (Sia) of GBS CPS. Binding amino acid will be analysed by Site-directed mutagenesis. Finally, Real-Time PCR array will be established and used to analyze expression levels of the genes including those in the immune response pathways, as well as inflammation cellular molecules related to immune response by the engagement of Siglecs by Sia of GBS. KEGG and GenMAPP Pathway annotation will be used to find the significantly differently expressed genes and the locations of these genes in immune response pathway. Research of this project will help to understand the interaction of pathogen and host between tilapia and GBS, and will provide target genes for preparation of vaccines, or candidate genes for molecular assisstant selection or transgenic research in order to breed new strains with high disease-resistant capacity, and will add more knowledge of molecular origination and evolution history of Siglecs.

2009年以来链球菌感染导致养殖罗非鱼大面积死亡，严重威胁着我国罗非鱼养殖业健康发展。本项目组在前期研究中确定了病原菌为无乳链球菌（GBS），且与感染人的主要GBS具相同分子血清型与序列分型。本项目拟结合分子生物学与免疫学实验技术，开展罗非鱼Siglecs分子研究，从嗜中性粒细胞中分离具抑制性ITIM结构域的Siglecs，通过受体与配体的结合, 确认识别GBS唾液酸的Siglecs位点、嗜中性粒细胞与GBS唾液酸的特异结合，点突变确定结合关键位点。并建立Real-Time PCR芯片方法检测唾液酸与Siglecs的结合对下游免疫信号通路主要因子、炎症细胞分子基因表达影响，KEGG和GenMAPP Pathway注释分析表达变化的基因及在信号通路上的定位。本研究将有助于对罗非鱼与GBS相互作用分子机制的了解，可望为疫苗研制、分子标记辅助育种及转基因培育抗病罗非鱼提供靶标、候选基因或新思路。

自2009年起GBS感染给中国养殖罗非鱼产业造成了重大的损失，对罗非鱼GBS 感染的分子机理了解的不足，成为科学有效防控链球菌病的瓶颈。Siglecs是一类能够识别唾液酸的最大的免疫超家族受体，哺乳动物的研究表明GBS可通过其荚膜上唾液酸多糖与宿主免疫细胞Siglecs相互作用，逃避宿主免疫。但鱼源GBS是否也能与宿主Siglecs互作实现免疫逃避尚不清楚。本研究首先通过FITC-GBS与罗非鱼嗜中性粒细胞的结合实验，确定嗜中性粒细胞表面存在与GBS结合的受体。结合转录谱分析、从粒细胞中扩增筛选到3个Siglecs基因，Siglec-1/-4b/-14 like。它们均具有与哺乳类Siglecs相似的基本结构、特异性结构域以及唾液酸多糖结合位点。Siglec-1/-4b带有免疫抑制型结构域，而Siglec-14带有免疫激活型结构域。通过构建Siglecs真核表达载体、转染获得Siglecs like/COS-7细胞及Siglecs/Ex-Fc融合蛋白。通过转染细胞与FITC-GBS的流式实验、融合蛋白与FITC-GBS的ELISA结合实验，证实Siglecs均具有较高的与GBS的结合活性。通过Siglecs/COS-7细胞与血细胞的凝集实验、融合蛋白与唾液酸多糖的ELISA结合实验，进一步证实了3个Siglecs均可与唾液酸多糖结合；qPCR检测在尼罗罗非鱼各组织的表达水平，Siglecs在脾、鳃与肾均有较高表达量；感染GBS后，Siglec-1/-14在肝、脾、肾及鳃组织都表现为前期显著上调后下调的趋势；而Siglec-4b在大部分组织的表达均为下调。在下游免疫炎症因子的检测中，抑炎因子在肝、脾、肾及鳃组织都表现为明显上调的趋势；促炎因子在脾、鳃组织都表现为前期显著上调后下调的趋势。在细胞水平上，检测转罗非鱼Siglecs/ RAW264巨噬细胞感染GBS后下游炎症因子的表达变化，发现促炎因子的表达上调和抑炎因子的表达下调也均受到抑制。研究结果表明，罗非鱼3个Siglecs分子均能识别GBS表面唾液酸多糖；感染GBS导致免疫相关组织Siglec表达水平的显著变化，且GBS与Siglecs的结合抑制了宿主下游免疫反应。此外还开展了罗非鱼抗菌肽的克隆表达、抗病转基因罗非鱼的培育、GBS PLGA微球口服疫苗的研制和温度对GBS唾液酸含量和毒力关系等相关研究。