Proteomics of the Plasmodium Ookinete

项目描述（由申请人提供）：本项目将应用蛋白质组学的先进和新兴技术，发现阻断疟疾传播的新分子靶点。虽然主要是探索性的，但这一建议也基于两个假设：1)疟原虫合子（蚊子中肠配子融合/受精的产物）表达新的，可能是低丰度的，但迄今为止尚未确定的蛋白质，可能是阻断疟疾传播的免疫靶点；2)眼动质疟原虫（继受精卵后主动侵入中肠的运动发育阶段）表达新的分泌和细胞表面相关分子，这些分子特异性参与寄生虫入侵蚊子中肠上皮，是阻断疟疾传播的潜在靶点。具体目的有两个：1)利用多维蛋白鉴定技术（MUDPIT）、定量同位素编码亲和标签质谱技术和定量二维凝胶电泳技术的互补技术，对鸡疟原虫受精卵和卵母细胞的蛋白质组进行描述，并利用生物信息学工具对现有疟原虫基因组数据库中受精卵和卵母细胞阶段基因进行比较鉴定，特别是在致死性人疟原虫恶性疟原虫中；2)利用新的DNA疫苗技术和传统的重组蛋白表达/疫苗接种，通过在传播阻断免疫动物模型中测试一些新鉴定的分子，启动合子/卵母细胞表达蛋白的验证。数据将被放入PlasmoDB公共数据库中。该申请与亲本R01授权AI45999“阻断疟疾传播的分子靶标”直接相关，因为当前申请的重点是使用蛋白质组学来发现和验证阻断疟疾传播的新分子靶标。这一提议也很适合由母体R01基金支持的现有的强大基础设施，用于测试新发现的寄生虫分子作为阻断疟疾传播的目标。这一应用与R21机制的探索性/开发性是一致的，因为它提出了应用新兴技术来发现未知但潜在重要和有趣的疟原虫分子。

DESCRIPTION (provided by applicant): This project will apply advanced and emerging technologies in proteomics to the discovery of novel molecular targets of blocking malaria transmission. While primarily exploratory, this proposal is also based on two hypotheses: 1) Plasmodium zygotes (the products of gamete fusion/fertilization in the mosquito midgut) express novel, perhaps low abundance, but hitherto unidentified proteins that may be immunological targets of blocking malaria transmission; and 2) Plasmodium ookinetes (the motile developmental stage that succeeds the zygote and actively invades the midgut) express novel secreted and cell surface-associated molecules that are specifically involved in parasite invasion of the mosquito midgut epithelium and are potential targets of blocking malaria transmission. There are two Specific Aims: 1) To delineate the proteome of Plasmodium gallinaceum zygotes and ookinetes using complementary techniques of multidimensional protein identification technology (MUDPIT), quantitative isotope-coded affinity tag mass spectrometry, and quantitative 2D gel electrophoresis, and use bioinformatics tools to comparatively identify zygote and ookinete stage genes in the extant Plasmodium genome databases, particular in the lethal human malaria parasite P. falciparum; and 2) To initiate the validation of zygote/ookinete-expressed proteins by testing a select number of newly identified molecules in an animal model of transmission-blocking immunity, both with a new DNA vaccine technology and traditional recombinant protein expression/vaccination. Data will be placed into the PlasmoDB public database. This application directly relates to the parent R01 grant AI45999, "Molecular Targets of Blocking Malaria Transmission," since the focus of the present application is on using proteomics to discover and validate new molecular targets of blocking malaria transmission. This proposal also fits well into the robust extant infrastructure supported by the parent R01 grant to test newly identified parasite molecules as targets of blocking malaria transmission. This application is consistent with the exploratory/development nature of the R21 mechanism since it proposes to apply new and emerging technologies to discovering unknown but potentially important and interesting Plasmodium molecules.