血吸虫正常发育雌虫与发育阻碍雌虫差异表达蛋白分析揭示雌虫生殖发育机理的研究

Schistosomiasis is a globally important helminthic disease of humans and animals, and is the second most common parasitic disease after malaria. In China, schistosomiasis still is a urgent public health issue despite the treatment has been available for several decades..Schistosome parasites are muticullular euaryotic organisms with a complex life cycle. Schistosomes have evolved separate sexes, eggs produced by the mature female worms are important in transmission of the parasite and responsible for pathogenesis. And the female schistosomes from singlesex infections show distinct differences from females obtained from bisexual infections. Females from singlesex.infections are underdeveloped in that they are stunted and exhibit an immature reproductive system. The singlesex infection can not injury the host too much for they can’t produce eggs and also don’t transmission the parasite.The studies on the difference between the singlesex infected females and bisexual infected females may give some clues in the mechanism of female worm’s reproductive development..In this study, the iTRAQ(isobaric Tagging for Relative and Absolute Quantitation)technique will be used to analysis the proteome difference between females from singlesex infections and females from bisexual infections in different developmental stages. Based on the bioinformatics about the difference, the factors which may be deeply involved in the reproductive developmental mechanism of female schistosome will be looking and studied in detail. The Real-time PCR, RNA inference, immunolocation, genechip, yeast two hybrid and Co-IP techniques will be used. Based on the importance of eggs, elucidate the reproductive developmental mechanism of female worms is important not only in.science, also in disease control and prevention.

血吸虫雌雄异体，成熟雌虫所产虫卵是造成宿主病理损害和疾病再传播的根源。而单性感染雌虫因其生殖器官处于发育阻碍状态，不能正常产卵，既不会对宿主造成严重的病理损害，也不会对血吸虫病的流行造成太大的影响。 因此， 从分析正常发育雌虫与发育阻碍雌虫间差异表达蛋白的研究入手来探索血吸虫雌虫生长发育，性成熟产卵的机理，可为阐明血吸虫生殖发育机理提供有价值的理论依据。本课题拟利用iTRAQ质谱定量技术分析不同发育时间节点正常发育雌虫与发育阻碍雌虫间蛋白质的表达差异，通过分析差异表达蛋白在生长发育过程中的表达趋势，并利用Real-time PCR、 RNA干扰、免疫组化、酵母双杂交和免疫共沉淀等技术，寻找并深入研究影响血吸虫雌虫生殖发育的分子，解析血吸虫雌虫发育调控机制，为研制发展高效抗血吸虫传播疫苗和新型病理阻断途径及抗血吸虫病药物筛选开拓新途径。

血吸虫雌雄异体，雌雄虫间的持续合抱是血吸虫雌虫生殖成熟的关键，成熟雌虫所产虫卵是造成宿主病理损害和疾病再传播的根源；单性感染雌虫处于发育阻碍状态，不能正常产卵，既不会对宿主造成严重的病理损害，也不会对血吸虫病的流行造成太大的影响。本研究利用iTRAQ结合质谱技术对18、21、23、25天正常发育雌虫和发育阻遏雌虫的蛋白表达情况进行了分析，共鉴定到了19423条肽段，3953个蛋白质；对四个发育时间节点的虫体蛋白进行了差异表达分析，利用MRM方法，Real-Time PCR 方法对差异蛋白质组学的结果进行了验证，且发现差异表达蛋白SjLGL在酵母体系和细胞水平上与血吸虫雌雄虫间信息传递相关蛋白SjGCP间存在相互作用；生物信息学分析表明随着血吸虫的生长发育，在正常发育雌虫中较多的蛋白参与合成与代谢，翻译修饰，蛋白质折叠和代谢进程，在发育阻遏雌虫中较多的蛋白参与生物体运动和细胞周期，氧化还原反应，能量代谢。进一步对差异表达蛋白SjLwr、SjFrzb2、SjELAV-like 1、SjELAV-like 2进行了生物学功能研究，结果发现：这4个蛋白在发育不同时间点的雄虫体内保持稳定，其在雌虫体内的表达则随着雌虫的发育呈变化态势，说明其与血吸虫雌虫的发育密切相关；长期干扰血吸虫体内相应蛋白的表达造成虫体存活能力，雌虫产卵能力，所产卵胚活力的大幅下降，进而减轻了宿主肝脏的虫卵负荷；相关蛋白被干扰会造成血吸虫形态学上的明显改变，如血吸虫虫体体表规律性结构的丧失和改变，血吸虫体内生殖系统相关的形态改变等。这些研究表明，这些差异表达蛋白可能用于开发免疫和/或小分子的治疗来控制血吸虫病的繁殖和传播的目标。