鱼类新母源性免疫因子的分离、鉴定与作用机理

Most fish eggs are fertilized externally, and the resulting embryos are exposed to an aquatic environment full of potential pathogens capable of causing various types of diseases. During the early stages of development, fish embryos have little or only limited ability to synthesize immune molecules endogenously and their lymphoid organs are not yet fully matured. How do they survive the pathogenic attacks in such a hostile environment? Fish eggs in most cases are cleidoic, i.e. closed free-living system following fertilization; they are thus supposed to depend on the maternal provision of immune molecules for protection against invading pathogens. Maternal immunity refers to the immunity transferred across the placenta, colostrum, milk or eggs from mother to offspring. It was first described in mammals and birds more than 110 years ago, and has now been documented among different vertebrates such as mammals, birds, reptiles, amphibians and fishes as well as in invertebrates including insect and amphioxus. Previous studies have shown that maternal IgM is able to be transferred from mother to offspring. Likewise, maternal transfer of innate immune factors including C3, lectins and lysozymes to offspring has also been reported in different teleost species. Additionally, immunization of parents results in a significant increase in IgM levels and anti-protease and lysozyme activities in their eggs compared to controls. These maternally-transferred molecules have been proposed to be involved in the early defense against pathogens in developing fish embryos. Using an in vitro assay system of complement activity, we have shown that the the protection of early embryos of zebrafish against microbial attack is attributed to maternal complement components operating via the alternative pathway. Despite these progresses in the past decade, many questions remain to be answered. For example, are there other factors in eggs also involved in maternal immunity? If so, how do they protect the embryos from microbial attack? What are their mode of action? To answer these questions also has a possible application in fisheries. Significant mortality is routinely recorded at larval stage, and how to enhance the survival rate of fish larvae is a problem of practical importance. The aims of this proposal were thus to isolate and identify the maternal immune molecules in fish eggs, to examine their functions and to explore their mode of action. We plan to use LPS-conjugated Sepharose chromotograpgy together with mass spectrometry to isolate and identify the maternal immune molecules in fish eggs. We will also utilize anti-microbial activity assay, protein-microbial interaction assay, ELISA, microbial challenge assay, microinjection, PCR, phagocytosis assay and scanning electronic microscope etc. to investigate the roles of these molecules in vitro and in vivo, and to uncover their mode of acion. For detailed research plan, see the flow chart in the proposal, please.

鱼类仔稚鱼和幼鱼死亡率通常很高，严重影响鱼苗生产。多数鱼类都是体外受精发育，其发育的水环境中存在大量致病微生物，而鱼类免疫系统发生较晚，胚胎不具备或仅有些许合成免疫分子的能力。鱼类胚胎在如此恶劣环境中，如何保护自己免受病原菌侵袭，是一个既有理论意义又有应用价值的重要科学问题。过去10多年，鱼类母源性免疫研究取得很大进展，但仍有大量问题悬而未决，如鱼卵中还有哪些新的免疫因子没有被发现？它们如何保护胚胎不受病原菌感染？它们作用机理是什么？作用能够持续多久？所有这些问题恰好构成本申请研究内容。我们拟以鲆鲽鱼为主要研究对象，利用交联LPS的琼脂糖凝胶或/和细菌与卵子提取液共育，分离结合蛋白，借助蛋白质质谱技术，实现对鱼类新的母源性免疫因子的高通量分离、鉴定，同时利用现代免疫学、微生物学和发育生物学等技术手段，研究它们对胚胎的保护作用和机理，取得具有原始创新性的新发现，为提高鱼苗成活率提供理论指导。

仔稚鱼和幼鱼高死亡率是鱼苗生产亟待解决的问题，多数鱼类都是体外受精发育，其发育的水环境中存在大量致病微生物，而鱼类免疫系统发生较晚，胚胎不具备或仅有些许合成免疫分子的能力，母源性免疫因作为提高鱼苗成活率的一条切实可行的途径并成为研究热点。但是，目前关于母源性免疫仍有许多问题悬而未决。本研究将斑马鱼早期胚胎匀浆上清液与交联有LPS的琼脂糖凝胶柱共育，分离、鉴定出了斑马鱼早期胚胎中的新型母源性LPS结合蛋白ZRANB2和ZNF365。首先，分别在分子水平和蛋白水平，证明了ZRANB2和ZNF365在斑马鱼各组织中均有表达，在胚胎发育早期ZRANB2和ZNF365也大量存在，这说明其确实为母源性蛋白。其次，体外功能验证证明了重组ZRANB2（rZRANB2）和重组ZNF365（rZNF365）作为模式识别受体可以识别细菌信号分子LPS，能与革兰氏阴性菌大肠杆菌、鳗弧球菌和嗜水气单胞菌结合，可以作为抗菌效应分子来直接杀死细菌。再次，还对其体内活性进行了验证，将rZRANB2和rZNF365用显微注射技术注入斑马鱼早期胚胎可以显著地提高胚胎对斑马鱼病原菌嗜水气单胞菌的抵抗能力，同时注入抗ZRANB2及rZNF365的抗体会让抵抗力明显地降低，而注入抗actin的抗体却没有此作用。最后，通过截短表达寻找到了ZRANB2和ZNF365的活性中心Z11/37和Z30-55，发现将其注入胚胎也会提高胚胎对嗜水气单胞菌的抵抗力。另外，我们在养殖鱼类大菱鲆的饲料中添加酵母葡聚糖（β-葡聚糖含量≥80%），80天后收集血液和卵子，制备血清和卵子提取液，发现其中溶菌酶的活性明显提高。说明酵母葡聚糖能够增强大菱鲆雌鱼血清和和卵子提取液的溶菌酶活性，从而提高亲代和子代的免疫能力。本研究对鱼类新的母源性免疫因子进行高通量分离、鉴定，探究其作用机理并致力于将其应用在鱼类养殖业中，对提高鱼类育苗成活率、促进我国鱼类养殖业特别是鲆鲽鱼类养殖业可持续发展有重要意义。