弹状病毒诱导斑马鱼皮肤和鳃免疫应答的蛋白质组学研究

项目名称： 弹状病毒诱导斑马鱼皮肤和鳃免疫应答的蛋白质组学研究

项目编号： No.31272692

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 吕爱军

作者单位： 天津农学院

项目金额： 80万元

中文摘要： 皮肤和鳃等组织器官是鱼类粘膜免疫系统的重要组成部分，但针对鱼类皮肤和鳃免疫应答的蛋白质组学，目前尚不清楚。在前期对斑马鱼皮肤免疫应答的转录组研究基础上，本项目拟采用鱼类弹状病毒浸泡感染的斑马鱼为实验动物模型，以皮肤和鳃为研究对象，进行比较蛋白质组学研究：1)采用双向凝胶电泳技术检测诱导前后皮肤和鳃组织的差异表达蛋白，并结合质谱鉴定和蛋白基因差异表达验证，应用差异蛋白质组学方法筛选皮肤和鳃免疫应答的关键蛋白/基因；2)构建诱导皮肤和鳃cDNA文库及关键基因诱饵表达载体，利用酵母双杂交技术筛选与之相互作用的蛋白，并进行生物信息学分析蛋白质相互作用网络，探索皮肤和鳃免疫应答信号通路的联系；3)通过诱导皮肤和鳃免疫应答关键蛋白的组织细胞定位及表达分析，在体内验证其功能。从分子、细胞和组织水平上研究斑马鱼皮肤和鳃免疫应答的蛋白质组学，旨在阐明鱼类粘膜免疫系统的调控机制，对鱼类病毒病防治具有重要意义。

中文关键词： 斑马鱼；皮肤；鳃；免疫应答；蛋白质组

英文摘要： Skin and gills are very important components of the mucosal immune system in fish, but the underlying molecular mechanism at the proteome-level remains unclear. In the present project, the zebrafish Danio rerio was used as an experimental animal model to study innate immune responses to the pathogenic rhabdovirus spring viremia of carp virus (SVCV) immersion infection based on our previous research experiences in mucosal immune responses of fish, and the proteomic analyses of both skin and gills in zebrafish will be performed as follows: Firstly, the profiling of the proteins present in the skin and gills of noninfected fish versus fish infected with virus was examined by using two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis, and the expression changes of the genes encoding the corresponding proteins were also evaluated by microarray and quantitative real-time PCR (qRT-PCR) analysis, in which the immune-related proteins and genes from the induced skin and/or gills were selected and confirmed by comparative proteomics method. Secondly, the bait protein expression vector and the cDNA library of the mixed tissues from the virus-induced skin and gill were respectively constructed, and the positive clones of prey protein were then screened by using the yeast two-hybrid system t

英文关键词： zebrafish；skin；gill；immune response；proteomics