刺激隐核虫诱发宿主产生保护性免疫反应的分子筛选

Cryptocaryon irritans is a parasitic ciliate of seawater teleost fish，causing deadly cryptocaryosis, which have been doing great harm to marine aquaculture in southeast coastal area. So far no safe and effective drugs to control this disease. It was reported that infection of C. irritans induced both innate and acquired immunities in the fish hosts. To screen for the molecules from C. irritans to induce host protective immunity, some related genes were cloned from a full-length cDNA library of C. irritans, their molecular characterizations were analyzed. The preliminary experiment showed that immunization with the recombinant proteins of some molecules protected the hosts against the challenge of C. irritans to a certain extent. In this study, the recombinant proteins or DNAs of the above mentioned molecules (such as papain-like cysteine protease, actin, profilin, and actin depolymerization factor) would be used to immunize Sebastiscus marmoratus or Trichinous ovatus, and Oreochromis niloticus × O. mossambicus，and the protective effect to the fish hosts would be observed and compared. Moreover, the molecular mechanism of the protective immunity would be clarified by detecting the fluctuations on the activities, expression levels of the factors involved in innate and acquired immunities. In addition, the molecular mechanism of the infection would be analyzed further by comparing the difference of immune-related factors between C. irritant-infected fish and untreated fish. Vaccine candidates with good protective effect to host fish would be expected.

刺激隐核虫是可引起鱼群体性死亡的海水硬骨鱼的寄生纤毛虫，严重危害我国东南沿海的海水鱼类养殖，尚无安全有效的防治方法。该虫感染能诱发宿主的先天性和获得性免疫，因此申请人从刺激隐核虫的全长cDNA文库中克隆、表达和鉴定了一批相关基因，初步结果显示有的基因的重组蛋白免疫大黄鱼后对致死剂量的刺激隐核虫的攻击具有一定的免疫保护作用。本项目拟系统地对前期鉴定过的分子免疫褐菖鲉或卵形鲳鲹、盐化养殖的罗非鱼，观察不同分子或组合（主要有木瓜蛋白酶样半胱氨酸蛋白酶、肌动蛋白及其主要调控因子）诱导宿主保护性免疫的效果；比较来自不同表达系统的重组蛋白和DNA疫苗的免疫效果差异；通过测定先天性和获得性免疫相关因子的活性大小、表达水平高低，阐明诱发免疫保护的分子机制。同时通过对刺激隐核虫幼虫感染组和未处理组的差别，进一步阐明刺激隐核虫感染的分子机制。期望能筛选出有较好免疫保护效果的疫苗候选分子。

刺激隐核虫（Cryptocaryon irritans）可寄生于海水硬骨鱼引起鱼群体性死亡，严重危害我国东南沿海的海水鱼类养殖，尚无安全有效的防治方法。以往研究表明该虫感染能诱发宿主的先天性和获得性免疫。本项目从C. irritans滋养体的cDNA文库中克隆、表达和鉴定了编码木瓜蛋白酶样半胱氨酸蛋白酶（CiPCP）、前纤维蛋白（CiProfilin）、表面蛋白（CiSA）、肌动蛋白解聚因子1（CiADF1）及肌动蛋白（CiActin）等多种基因；用重组蛋白rCiPCPt、rCiADF1和rCiSA或重组的真核表达载体pcDNA/CiPCP、pcDNA/CiPCPt、pcDNA/CiProfilin、pcDNA/CiActin、pcDNA/CiPCPt-CiProfilin、CiProfilin-CiActin等免疫大黄鱼，观察它们是否有助于大黄鱼抵御亚致死剂量的C. irritans攻击；用Real-time PCR测定了免疫后的大黄鱼中IL-1β、MHCII、Myd88、TLR-8、IL-8A等基因的表达水平与未免疫组的差异；分别用大黄鱼的鳍条、脑髓、褐菖鲉的鳍条、海洋青鳉鱼的鳍条等组织原代培养、继而传代培养，建立了5个细胞系；这些细胞均能表达以pcDNA为载体转染的外源基因；免疫实验的结果显示：重组蛋白rCiPCPt、rCiADF1和rCiSA免疫组大黄鱼的相对免疫保护率分别为41%、59%和50%；而注射质粒DNA对大黄鱼的相对保护率分别是44.5%（pcDNA/CiPCP）、37.9% （pcDNA/CiPCPt）、40.9% （pcDNA/CiPCPt-CiProfilin） 、49%（pcDNA/CiProfilin）、36.3%（pcDNA/CiSAt）、52%（pcDNA/CiActin）、28%（CiProfilin-CiActin）、9%（pcDNA）。免疫相关基因表达情况的检测结果中较有共性的是免疫组的样品中IL-1β的上调。另外还用海洋青鳉鱼建立了C. irritans感染的小动物模型。本项目将为刺激隐核虫病的病原生物学的进一步研究奠定基础，也为防治方法的开发提供参考。