ISCOMs疫苗对斑马鱼非特异性免疫的调节

Our study is the first time to use ISCOMs vaccine in zebrafish, complete the zebrafish immune organ morphology, analysis of the number of macrophages, neutrophils and other immune cells and functional changes. Substractive cDNA library was constructed by suppression subtractive hybridization (SSH) in zebrafish vaccinated by ISCOMs vaccine. The clones are selected randomly from the library, then screened by PCR identification, dot blot hybridization and DNA sequencing, after bioinformatic analysis the genes related to innate immunity are selected. Using real-time PCR method to further validate selected the difference significantly candidate genes, then some of the genes are cloned and analysis the function. The ISCOMs vaccinated zebrafish are challenged by vibrio vulnificus, and aeromonas hydrophila, and then gene functions are study in vivo. In summary, our preliminary study is trying to understand how ISCOMs vaccine through the regulation of gene expression in the innate immune system, change the function of the immune cells to resist pathogen invasion mechanism. This study will provide the basis of theoretical research for using ISCOMs vaccine in fish, and give new ideas for the use of aquatic vaccine.

本研究首次以ISCOMs疫苗免疫斑马鱼，观察免疫后斑马鱼免疫器官的形态改变，分析巨噬细胞、中性粒细胞等免疫细胞数量及功能的变化。通过构建疫苗免疫后差异表达基因的抑制性差减杂交文库，对该文库中随机挑取的克隆经PCR鉴定、斑点杂交筛选和DNA测序，进行生物信息学分析，筛选出非特异免疫系统相关基因。采用real-time PCR方法进一步验证，选出差别显著的候选基因，进行基因克隆和功能分析。用创伤弧菌、嗜水气单胞菌对ISCOMs疫苗免疫后的斑马鱼攻毒，体内检测相关基因的功能。旨在初步了解ISCOMs疫苗通过调节非特异性免疫系统中的基因表达，改变免疫细胞的功能，抵御病原菌入侵的作用机理。该研究可为ISCOMs疫苗在鱼类中的应用提供理论基础，为水产疫苗的选择和应用提供新的思路。

近海养殖业是我国海洋经济的主要组成部分，近年来发展迅速，但随着养殖规模的扩大，病害也日渐增多。本团队研制的嗜水气单胞菌、创伤弧菌ISCOMs疫苗目前已经应用到鳗鲡和大黄鱼等鱼类的病害防治中。实际应用中发现仅包含牛血清白蛋白（BSA）的ISCOMs就能提高鱼类基础免疫水平，诱导有效的免疫保护。ISCOMs疫苗对鱼类非特异性免疫调节的机理还不清楚，国内外尚未见ISCOMs疫苗免疫斑马鱼的报道。.本研究建立了ISCOMs疫苗免疫斑马鱼的模型，确定ISCOMs疫苗免疫的安全剂量、时间、次数及免疫方式。ISCOMs疫苗免疫斑马鱼后，用创伤弧菌进行攻毒后免疫组存活率显著高于对照组，证明疫苗具有保护效力。观察比较创伤弧菌及嗜水气单胞菌感染及疫苗免疫后免疫器官的形态变化。构建疫苗免疫前后差异表达基因的抑制性差减杂交文库，结合RNA测序技术，通过生物信息学分析筛选出非特异免疫系统相关基因。发现ISCOMs疫苗免疫后只有极少数基因表达发生变化，但免疫后病原菌攻毒后可引起大量基因表达变化。ISCOM疫苗免疫前后则只有12个基因表达有变化，创伤弧菌感染前后有295个基因表达变化，但是ISCOM疫苗免疫后创伤弧菌攻毒则有高达772个基因有差异表达。嗜水气单胞菌感染前后有127个基因表达变化，疫苗免疫后嗜水气单胞菌攻毒有414个基因改变。采用real-time PCR方法对部分基因进行检测，克隆了重要的炎症相关因子TNF（肿瘤坏死因子）并开展相关功能研究。初步了解斑马鱼非特异性免疫系统的变化规律及抵御病原菌入侵的作用机理。本研究可为ISCOMs疫苗在鱼类中的应用提供理论基础，为水产疫苗的选择和应用提供新的思路。