嗜水气单胞菌感染对青鱼补体系统激活的影响机制研究

The black carp Mylopharyngodon piceus is the culturally important domestic fish used in polyculture in China. In recent years, the disease caused by Aeromonas hydrophilia in black carp outbreak frequently. A. hydrophilia is an important fish pathogen that has been known to cause septicaemia, ulcerative and haemorrhagic diseases primarily in freshwater fish, which lead to huge economic loss. Basing on the previous study，quantitative proteomics of serum and liver from healthy black carp and black carp infected by A. hydrophilia will be studied using iTRAQ in the project, and the different expressions of protein from two samples will be quantified to discuss the interaction between black carp and A. hydrophilia. Then, the proteins (complement component 9, complement factor B, etc.) closely related to activation of complement system of black carp will be selected from the differentially expressed proteins. Then, the full length cDNA sequence of complement component 9 and factor B will be cloned by RACE method, and the changes in gene and protein expression of these two complement components in black carp during infection of A. hydrophilia will be investigated by quantitative real-time RT-PCR and Western blot respectively. After implementation of the project, the influence mechanism of A. hydrophilia infection on complement system activation in black carp will be explored firstly, which also may contribute to clarify the immune escape mechanism of A. hydrophilia and enrich the knowledge of fish immunology.

青鱼是我国淡水养殖四大家鱼之一，近年来随着养殖产量增加，病害问题不断出现，其中以嗜水气单胞菌（Aeromonas hydrophilia）引起的病害较严重。本项目拟采用iTRAQ蛋白质组学方法分析嗜水气单胞菌感染组和对照组青鱼的血清和肝脏，筛选、鉴定差异表达蛋白，并从中筛选出与补体系统激活相关的组分（前期预实验已筛选出补体C9和补体B因子）。用RACE方法进一步克隆补体C9、补体B因子等基因cDNA全长，实时定量PCR检测感染过程中补体C9和B因子转录水平的表达变化。然后通过原核表达载体构建、表达及抗体的制备，Western blot分析感染过程中补体C9和B因子蛋白水平的表达变化。目前关于嗜水气单胞菌感染对鱼类补体系统激活的影响机制研究，尚未见相关报道。本项目的开展，对阐明嗜水气单胞菌感染对鱼类补体系统激活的影响机制，充实鱼类免疫学的基础数据及有效防控鱼类病害具有较大的理论和指导意义。

青鱼是我国淡水养殖四大家鱼之一，近年来随着养殖产量增加，病害问题不断出现，其中以嗜水气单胞菌（Aeromonas hydrophila）引起的病害较严重。本项目采用iTRAQ结合LC-MS/MS技术对嗜水气单胞菌感染组和对照组青鱼的血清和肝脏开展了蛋白质组学研究，筛选、鉴定青鱼感染嗜水气单胞菌后的差异表达蛋白。KEGG通路分析表明，血清和肝脏中的差异表达蛋白均在补体和凝血级联反应通路富集最为显著，其中包括补体C1、补体B/C2A、补体B/C2B、补体C3、补体C4等，这些补体蛋白参与了补体活化的3条激活途经（经典途径、替代途径和凝集素途径），在青鱼感染嗜水气单胞菌后均呈明显下调表达，提示细菌感染抑制了补体各激活途经，鱼体免疫防御机能下降。采用实时荧光定量PCR方法，从转录水平分析了青鱼感染嗜水气单胞菌后不同组织中补体B/C2A、补体C3和补体C9基因表达的变化，结果显示其转录水平与蛋白水平的变化并不完全一致。制备了补体C3的抗体，Western blot分析表明补体C3在血清中的含量最高，青鱼感染嗜水气单胞菌后，补体C3在心脏中的表达下调。免疫荧光组织化学分析结果显示，青鱼感染嗜水气单胞菌后，补体C3在肾脏中的表达明显增强。此外，从组织病理学角度阐述了嗜水气单胞菌感染对青鱼不同组织造成的损伤，尤其是肾脏的损伤与上述补体在青鱼感染嗜水气单胞菌后的下调表达密切相关。本项目的研究结果，对阐明嗜水气单胞菌感染影响鱼类补体系统激活的机制，进一步解析嗜水气单胞菌的致病机理以及其逃避宿主补体攻击的机制，对充实鱼类免疫学的基础数据、有效防控鱼类病害具有一定的理论和指导意义。