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

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 to identify the interacting proteins, which of these interactions were further analyzed by bioinformatics methods, thus the possible network of signaling pathways involved in skin and gills immune responses were explored at the proteomic and cellular levels. Thirdly, the expression and cellular localization of the bait and prey proteins were assessed in the skin and gills tissues using Western blot and immunohistochemistry (IHC) analysis, and the prey proteins were confirmed to interact directly with the bait protein in vivo. Taken together, the proteomic analysis of the immune response of zebrafish skin and/or gills were performed at the molecular, cellular and tissue levels, and thus the antiviral defense mechanisms of skin and gills in fish were elucidated in detail. The results from this work will contribute to the understanding of the molecular regulatory mechanism of the mucosal immune system, which have important scientific and practical significance for the prevention and treatment of viral diseases in fish.

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。

鱼类鳃和皮肤黏膜免疫系统在机体抗感染防御中发挥重要作用。本项目采用双向凝胶电泳(2-DE)结合基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术研究病原感染诱导斑马鱼(D. rerio)皮肤免疫应答的蛋白质组学，通过同位素标记相对和绝对定量(iTRAQ)、高效液相色谱-串联质谱(LC-MS/MS)技术等进行斑马鱼鳃免疫应答的差异蛋白质组分析。结果表明，在斑马鱼鳃中鉴定获得1338个蛋白，主要包括细胞过程(15.36%)、代谢过程(11.95%)和生物调控(8.29%)相关蛋白，进一步筛选获得鳃差异表达蛋白82个，其中有57个上调表达蛋白和25个下调表达蛋白。根据基因蛋白功能聚类(GO)分析表明鳃中33个差异表达蛋白(8.8%)参与应激与免疫应答过程，其中上调表达蛋白主要有补体、丝氨酸蛋白酶肽酶抑制因子、膜联蛋白和组蛋白等，KEGG通路分析表明补体和凝血级联、致病性大肠杆菌感染以及吞噬体等信号通路参与鳃抗感染免疫应答机制。在感染诱导斑马鱼皮肤检测到1000多个蛋白点，鉴定获得17个皮肤免疫相关蛋白，其中6个上调表达蛋白，11个下调表达蛋白，主要包括免疫球蛋白重链可变区、主要组织相容性复合体、甘油醛-3-磷酸脱氢酶、AP2转录因子、KRT4蛋白以及4个未知功能蛋白等。通过Illumina Hiseq2000高通量测序结合实时荧光定量PCR（qRT-PCR）技术进行鲫皮肤和鳃免疫转录组学分析，结果获得鲫皮肤免疫相关基因94776个、鳃84259个，其中皮肤C反应蛋白（CRP-2）、主要组织相容性复合体（MHC-Ι、MHC-II）、补体（C7）、白细胞介素（IL-22），鳃MHC-I基因、白细胞介素（IL-17C）等基因显著性差异表达，主要发现有抗原加工和呈递、吞噬小体、细胞粘附分子、趋化因子、补体和凝血系统、NK细胞介导细胞毒作用、细胞凋亡、TLR、NF-κB、BCR、TCR、NLR、JAK-STAT、MAPK、TGF-β、Wnt、VEGF、Notch和mTOR等信号通路参与皮肤和鳃黏膜免疫应答过程。在蛋白质水平研究发现感染鲫皮肤中肌动蛋白、肌酸激酶M型、热休克蛋白同源71kDa等参与免疫应答过程等。本研究首次初步揭示病原感染诱导斑马鱼、鲫鱼皮肤和鳃蛋白质组学及其基因蛋白网络调控，不仅有助于理解鱼类黏膜免疫应答的分子机制，而且为鱼类疾病防治提供科学理论依据。