鳜源嗜水气单胞菌菌蜕投递传染性脾肾坏死病毒抗原研究

We have proved that immune cells distributed in mucosa-associated lymphoid tissues provide cell foundation for mucosal immune response in mandarin fish. So immersion adminstration is the most convenient inoculation routes for vaccination of mandarinfish fingerlings groups. However it needs massive dose of antigen by immersion inoculation routes and it is unacceptable at cost for killed virus vaccine or subunit vaccine. What is the cheap delivery vector? Our preliminary studies had proved that bacterial ghost can elicit strong mucosal immune response in fish and has characteristic of immune adjuvant and targeted APC. Therefore, bacterial gost could be a good delivery vector candidate for viral antigen. In this study, we will use mandarin fish co-infected by infectious spleen and kidney necrosis virus (ISKNV) and Aeromonas hydrophila (Ah) as an animal model, and display the antigen gene of ISKNV on the surface of Aeromonas hydrophila ghost by homologous recombination technology to obtain a kind of recombinant ghost expressing ISKNV antigen. Then the level of immune response of the recombinant ghost vaccine will be evaluated to explore the possibilities of delivery viral antigen on Aeromonas hydrophila ghost by immersion administration. This study will provide a kind of cheap, convenient, and efficient delivery vector of immersion adminstration for mandarin fish vaccine, and provide a new idea for resolving the difficulty of no convenient and efficient immersion delivery system for virus vaccine in fish.

我们已经证明鳜鱼局部粘膜具有完成免疫应答的细胞基础（见研究基础），基于粘膜免疫的浸泡免疫途径成为鳜鱼苗种群体免疫最为实用的接种方式。然而浸泡免疫所需抗原量大，对于病毒灭活疫苗或亚单位疫苗来说成本上无法接受。如何获得病毒抗原廉价投递载体呢？前期试验结果显示，细菌菌蜕能引起鱼类强烈的粘膜免疫应答，同时兼具免疫佐剂、抗原呈递细胞靶向性等免疫特性，是一种潜在的鱼类病毒抗原投递载体。因此，本研究以并发感染嗜水气单胞菌（Aeromonas hydrophila,Ah）及传染性脾肾坏死病毒（ISKNV）的鳜鱼为模型，通过同源重组技术将ISKNV抗原呈递于Ah菌蜕表面，获得表达ISKNV 抗原重组Ah菌蜕，评价重组菌蜕引起鳜免疫应答水平，探讨菌蜕通过浸泡免疫投递病毒抗原可行性。本研究的成功将提供一种低成本、简便、高效的鳜鱼疫苗浸泡免疫投递载体，为解决鱼类病毒疫苗浸泡投递系统难题提供了一种新的思路。

本项目针对由嗜水气单胞菌(Aeromonas hydrophila，Ah)及虹彩病毒（传染性脾肾坏死病毒，ISKNV）并发感染造成养殖鳜鱼暴发性死亡的严重问题，构建了简便、高效鳜重组嗜水气单胞菌、虹彩病毒病二联菌蜕疫苗。通过生物信息分析筛选获得ISKNV的MCP、NP、EP等3个候选抗原，成功构建了候选抗原的原核表达载体及真核表达载体，并实现高效表达。这些候选抗原重组蛋白和质粒DNA制备的疫苗分别免疫鱼体后进行攻毒，发现EP蛋白免疫效果最佳，确定EP蛋白为ISKNV保护性抗原用于重组疫苗的构建。接着采用基因组步移的方法扩增获得嗜水气单胞菌ompA 基因序列，通过同源重组将ISKNV EP基因同源重组至嗜水气单胞菌菌体表面ompA第三外环中，构建了在嗜水气单胞菌菌体表面融合表达ISKNV EP抗原的重组菌株，将已构建好的含噬菌体Lysis E基因的裂解质粒转入重组嗜水气单胞菌中，成功将传染性脾肾坏死病毒EP抗原呈递至嗜水气单胞菌菌蜕表面。通过测定重组疫苗免疫小鼠和鳜鱼后血清效价及淋巴细胞增殖指数，确定了该重组疫苗可以激发机体体液免疫和细胞免疫应答，免疫攻毒试验结果表明该重组疫苗对ISKNV注射免疫相对保护率为73.3%，浸泡免疫相对保护率为56.3%。本研究提供了一种低成本、简便、高效的鳜鱼嗜水气单胞菌展示ISKNV抗原重组疫苗，并开发出一种渔用疫苗浸泡免疫投递载体，为解决鱼类病毒疫苗浸泡投递系统难题提供了一种新的思路。