鱼卵中肝素结合蛋白的分离和功能分析：新的母源性免疫因子的鉴定

鱼类胚胎和仔稚鱼死亡率通常很高，严重影响鱼苗生产。多数鱼类胚胎在具有大量致病微生物的水中发育，而鱼类胚胎不具备或仅有些许合成免疫分子的能力，免疫系统发育、成熟又晚。它们在如此恶劣环境中如何保护自己不受病原感染呢？这是一个具有理论意义和应用价值的重要科学问题。过去近20年，鱼类母源性免疫研究取得长足进展，但仍有许多问题未解决，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感染？它们作用机理是什么？作用能持续多久？这些也正是本申请想解决的问题。大量研究表明多数肝素结合蛋白为抗菌肽。因此，我们拟利用肝素交联的琼脂糖凝胶与鲆鲽鱼卵子提取液共育，分离结合蛋白，借助蛋白质质谱和生物信息学技术实现对鱼卵中候选的新的抗菌肽的分离和鉴定，同时利用现代免疫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，取得具有原创性的新发现，揭示鱼类母源性免疫规律，为提高鱼苗成活率提供理论支撑。

Most fish eggs are fertilized externally, and thus their embryos/larvae 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/larvae have little or only limited ability to produce 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 following fertilization, and are thus supposed to depend upon 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 both the alternative and lectin pathways. Very recently, we have also shown that fish egg lecctin, chitinase, phsvitin and ZRANB2 are newly identified maternal immune factors, that all function in immune defense in fish embryos/larvae. Despite these progresses in the past decade, many questions remain to be answered. For example, how many uncharacterized immune factors are there in fish eggs? How do they protect the embryos from microbial attack? What are their mode of action? How long can their functions be maintained during development? Solution of these problems may have a possible application in fisheries. Significant mortality is routinely recorded at larval stage, and how to enhance the survival rate of fish larvae is an issue of practical importance. The purposes of this proposal are to isolate and identify the maternal immune molecules in fish eggs, to examine their functions and to explore their mode of action. Accumulating data have shown that many heparin-binding proteins are antimicrobial peptides. Therefore, we plan to use heparin-conjugated Sepharose chromotograpgy, combining with mass spectrometry, to screen uncharacterized maternal immune molecules present in fish eggs. We will also utilize anti-microbial activity assay, protein-microbial interaction assay, ELISA, microbial challenge assay, microinjection, PCR, phagocytosis assay, scanning electronic microscope and gene knockdown etc. to investigate the roles of these molecules both in vitro and in vivo, and to uncover their mode of action. For detailed research plan, see the flow chart in the proposal, please.