基于四膜虫表达体系的刺激隐核虫保护性抗原的筛选和鉴定

Cryptocaryon irritans, an important marine ciliate protozoan that causes a disease referred to as "marine white spot disease", has already become one of the most harmful parasites in Chinese marine aquaculture industry. The vaccination is considered to be a reasonable mean to prevent the infection of C. irritans. However, lacking adequate protective antigens gradually become a conspicuous limiting factor to develop the efficacious vaccine of C. irritans. In this study, we will screen candidate protective antigens by the two dimensional immunoelectrophoresis from specific stage antigens of C. irritans, especially devote to find the antigen profile of the invasive stage and the cilia of the C. irritans. Meanwhile, we will construct the C. irritans expression library by the Tetrahymena thermophila expression system, and directly screen and identify candidate protective antigens from this library. Concrete operations are as follows: 1. To obtain respectively the full-length cDNA and suppression subtractive hybridization library of theront, trophont and cyst of the C. irritans. 2. Constructing the the obove genes into the T. thermophile anchoring expression vector which will make the recombinant proteins bound the membrane and cilia of T. thermophile. 3. To screen vaccine candidate antigens of C. irritans from T. thermophile anchoring expression system by the methods of Immunoblotting test and Immobilization assay. 4. To transform anchoring-expression recombinant transformant to the secretory-expression type, producing recombinant candidate antigens for the immune protection experiment. Above all, the results of this project will have great significance for promoting the vaccine development of C. irritans and bring the breakthrough in the study of immunological control of C. irritans.

刺激隐核虫病是我国海水鱼类的重要病害，给海水养殖业造成了巨大的损失，但目前尚无有效的防治措施。免疫接种已被证实可能成为刺激隐核虫病防控的有效方法，但对该虫免疫保护性抗原的相关研究尚极欠缺，也无法获得足够的抗原用于疫苗生产。本项目拟通过两条途径解决此关键问题，首先通过免疫蛋白组学方法，多层次比较刺激隐核虫各期虫体免疫抗原的差异性，突出显示幼虫期的侵染性功能抗原和纤毛抗原谱，分析其结构特点，获得期特异性抗原信息，对刺激隐核虫期特异性抗原进行筛选和鉴定；其次通过获得刺激隐核虫各期虫体的完全表达谱和差异表达谱基因，并与四膜虫微管蛋白基因重组，然后导入基因工程四膜虫中，构建四膜虫锚定型表达文库，从中筛选和鉴定出刺激隐核虫期特异性抗原基因和组分特异性抗原基因。最后利用四膜虫分泌型表达体系对刺激隐核虫抗原基因进行表达，获得大量保护性免疫抗原，为刺激隐核虫疫苗开发奠定基础。

免疫接种已被证实可能成为刺激隐核虫病防控的有效方法，但对该虫免疫保护性抗原的相关研究尚极欠缺，也无法获得足够的抗原用于疫苗生产。基于此本项目开展了刺激隐核虫保护性抗原的筛选与鉴定，具体研究内容以下：1. 通过测定刺激隐核虫生活史3个时期转录组，鉴定到2470个期特异性表达或高表达基因。其中滋养体和幼虫比较得到2011个差异表达基因，包囊和幼虫比较得到1404个差异表达基因，包囊和滋养体比较得到1797个差异表达基因；2. 通过比较蛋白组学，鉴定出86个显著差异表达蛋白点，属于44种特定蛋白质，主要包括微管蛋白、肌动蛋白、V型ATP合酶、烯醇化酶、Mcm2-3-5家族蛋白、26S蛋白酶体亚基P45家族蛋白和热激蛋白70等；3. 运用免疫蛋白组学技术，从3个发育时期共鉴定到84个和28个分别能与兔和鱼抗刺激隐核虫血清反应的蛋白点，质谱成功鉴定出87个蛋白点，对应20种蛋白，主要包括肌动蛋白、α-微管蛋白、β-微管蛋白、热激蛋白70、线粒体热激蛋白70、V型ATP合酶催化亚基α、dnaK等等；4.通过前面的研究，项目组获得了大量刺激隐核虫亚单位疫苗用候选蛋白基因。本项目利用四膜虫分泌型和粘液型表达体系，成功表达出了GDCI1重组蛋白，免疫石斑鱼后获得44%的相对保护率；5.研究了石斑鱼抗刺激隐核虫的免疫机制：发现NCCRP+细胞以及TLR信号通路参与抗寄生虫免疫应答。