Structure and functional analysis of high molecule weight rhoptry protein complex, RhopH, of rodent malaria parasite.

啮齿动物疟原虫高分子量棒状体蛋白复合物 RhopH 的结构和功能分析。

• 批准号: 14370084
• 负责人: TORII Motomi
• 金额: $ 8.96万
• 依托单位: Ehime University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370084/
• 关键词: infectious disease; malaria; Plasmodium; invasion; protein expression; RT-PCR; parasitology; メロゾイト

Invasive form of malaria parasite utilizes its apical organelle such as rhoptry for the erythrocyte invasion. One of the rhoptry molecules, RhopH complex, is known to bind to erythrocyte surface. To clarify the characteristic feature of the erythrocyte receptor and the binding domain of the RhopH complex, we undertook to generate a panel of recombinant proteins and antibodies against P. falciparum RhopH complex this year. Firstly, we modified expression plasmid by adding myc-tag and green fluorescent protein in order to make the downstream analysis easier. Secondly, we adapted Gateway system (Invitrogen) to make expression constructs easier, because without this technique, it was difficult to make the final large plasmid constructs. Thirdly, we divided the RhopH1 gene, which we thought a strongest candidate for the binding component in RhopH complex by a genetic analysis, into 3 parts and ligated into the expression constructs. Currently we are evaluating the expression from these constructs in the mammalian system.On the other hand, we successfully generated recombinant proteins in E. coli and obtained specific antibodies against each components of P. falciparum RhopH complex. We also obtained specific antisera by DNA immunization. These sera were found to be reactive against the parasite native proteins by immunofluorescence microscopy and Immunoblot analysis. These are currently used for immunoprecipitation of the RhopH complex, which bound to and eluted from erythrocyte-surface, to evaluate the character of the erythrocyte receptor.

疟原虫的侵袭性形式利用其顶端细胞器（例如棒状体）来入侵红细胞。已知棒状体分子之一 RhopH 复合物可与红细胞表面结合。为了阐明红细胞受体的特征和 RhopH 复合物的结合域，我们今年致力于生成一组针对恶性疟原虫 RhopH 复合物的重组蛋白和抗体。首先，我们对表达质粒进行了修饰，添加了myc标签和绿色荧光蛋白，以便于下游分析。其次，我们采用了 Gateway 系统（Invitrogen）以使表达构建体更容易，因为如果没有这种技术，就很难制作最终的大质粒构建体。第三，我们将 RhopH1 基因（通过遗传分析认为是 RhopH 复合物中结合成分的最强候选者）分成 3 部分，并连接到表达构建体中。目前我们正在评估这些构建体在哺乳动物系统中的表达。另一方面，我们成功地在大肠杆菌中产生了重组蛋白，并获得了针对恶性疟原虫RhopH复合物的每种成分的特异性抗体。我们还通过DNA免疫获得了特异性抗血清。通过免疫荧光显微镜和免疫印迹分析发现这些血清对寄生虫天然蛋白具有反应性。这些目前用于 RhopH 复合物的免疫沉淀，该复合物与红细胞表面结合并从红细胞表面洗脱，以评估红细胞受体的特性。

项目成果

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