克氏原螯虾不同类型血细胞在感染螺原体前后的iTRAQ定量蛋白质组学分析

Spiroplasma eriocheiris is a novel pathogen causing systemic infection in the crayfish Procambarus clarkii. Hemocytes are the primary infection targets of S. eriocheiris and also the most important immune cells in the crustacean cellular defense system. This project will be based on our primary results of the classification and in vitro culture of the hemocytes from P. clarkii and transcriptome sequencing of the crayfish. We intend to do further research about the quantitative proteomic analysis of P. clarkii hemocytes upon S. eriocheiris infection. Firstly, we will separate the different sub-populations of the hemocytes and cultured them in vitro separately, using the Fluorescence-Activated Cell Sorter (FACS). Secondly, we utilized a quantitative proteomics strategy that combined 8-plex isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) to obtain the protein reference maps (PRM) and identify specific molecular signatures of each subpopulation of the hemocytes from P. clarkii. What's more, by comparing with the PRM of each hemocyte type after S. eriocheiris infection, we could also develop predictive markers and analysis the different immune functions of each hemocyte type. Finally, some of the important different expressed proteins which are potentially the key proteins in the pathogen-host ineraction will be selected to carry out further research to provide novel insights into the molecular mechanisms underlying the innate immune system. Our results will not only demonstrate the utility of iTRAQ-based high-throughput quantitative proteomic analysis in pathogen-host proteomics research, provide novel hemocyte lineage markers, and reveal the regulatory functions of different kinds of hemocyte, but also represent the functional molecules during the S. eriocheiris infection immune processes.

螺原体是近年来发现的一种引起克氏原螯虾重大病害的新型病原微生物，而血细胞是其侵染的重要靶细胞，同时也是克氏原螯虾最重要的免疫细胞。本项目以克氏原螯虾血细胞的分类、体外培养及转录组测序为基础，以比较蛋白质组学分析为主线，动态、整体和定量地展示不同类型血细胞在感染螺原体前后的蛋白质表达谱，获得关键差异蛋白的定性、定量和功能信息。首先，结合流式细胞术分选和体外培养，实现不同亚群血细胞的分离与富集；然后，采用高通量的8重iTRAQ标记结合2D LC-MS/MS的定量蛋白组学技术，对接已有的转录组数据，全面展示各型血细胞在螺原体感染前后的蛋白表达谱，筛选各型血细胞的特异性分子标记及与螺原体感染相关的蛋白标记物，比较不同血细胞的蛋白表达模式差异；最后，挖掘可能与螺原体病的发生、发展相关联的重要调控靶点分子，研究其免疫调控功能。以上研究将为诠释虾蟹先天免疫防御的分子调控机理和建立疾病防治新对策奠定基础。

本项目围绕克氏原螯虾这一具有重要经济价值的水产养殖品种的螺原体重大病害，以血细胞为切入点，采用高通量的iTRAQ定量蛋白组学方法，系统研究了克氏原螯虾对螺原体的免疫防御反应，取得了系列研究结果，包括：. ①采用显微和超微结构观察和流式细胞仪分析等方法，系统研究了不同类型克氏原螯虾的形态特征：按照细胞大小、数量、颗粒度和核质比等特点，可以将克氏原螯虾血细胞区分成三种类型，分别为：大颗粒细胞（G）、小颗粒细胞（SG）和透明细胞（H）。进而围绕Grace’s insect media构建了血细胞的体外原代培养体系。. ②分别探索了流式细胞分选和Percoll密度梯度离心两种方法对不同类型克氏原螯虾血细胞的分离效果，检测了分离后血细胞的蛋白质量：流式细胞仪分选时，流动室内鞘液的无机盐、pH与渗透压等都对血细胞的离体存活产生较大影响；而密度梯度离心的样品存在一定蛋白污染，综合考虑后，选择从个体水平上开展后续研究。. ③采用高通量的定量蛋白组学方法从整体水平上研究了血细胞对螺原体感染的免疫防御特征，发掘了螺原体与宿主互作中的核心信号通路和关键免疫因子：采用转录组和蛋白质多组学联合分析的方法，对差异蛋白进行了较好地注释和分析，累计鉴定出蛋白1593种，成功定量出的为950种；其中螺原体感染前后的差异表达蛋白为285种，上调83种，下调202种，绝大多数发挥粘附和代谢调控功能，与螺原体的致病特征相吻合。这些结果为后续研究提供了关键依据。. ④结合个体感染模型，分析了克氏原螯虾血细胞在螺原体感染后的免疫特征及抗氧化系统关键酶活性变化，讨论了不同类型血细胞的免疫反应特征。. 另外在项目研究过程中，围绕克氏原螯虾混养池塘中的其它虾蟹的病毒和寄生虫病原也开展了研究，比较了克氏原螯虾等虾蟹对不同病原的免疫防御策略。