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.

侵袭型疟原虫利用其根尖细胞器如淋巴细胞进行红细胞侵袭。其中一种核糖核酸分子，核糖核酸复合体，已知与红细胞表面结合。为了明确红细胞受体和RhopH复合物结合域的特征，我们今年着手制作了一组针对恶性疟原虫RhopH复合物的重组蛋白和抗体。首先，我们通过添加myc标签和绿色荧光蛋白对表达质粒进行修饰，使下游分析更加容易。其次，我们采用了Gateway系统（Invitrogen）来简化表达构建，因为没有这种技术，很难构建最终的大质粒构建体。第三，通过遗传分析，我们认为RhopH1基因是RhopH复合体中结合组分最强的候选基因，我们将RhopH1基因分成3个部分，并连接到表达构建体中。目前，我们正在评估这些结构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生成了重组蛋白，并获得了针对恶性疟原虫RhopH复合物各组分的特异性抗体。我们还通过DNA免疫获得了特异性抗血清。免疫荧光显微镜和免疫印迹分析发现，这些血清对寄生虫的天然蛋白有反应。这些目前用于RhopH复合物的免疫沉淀，RhopH复合物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

Kawazu S, Nozaki T, Tsuboi T, et al.: "Expression profiles of peroxiredoxin proteins of the rodent malaria parasite Plasmodium yoelii."International Journal for Parasitology. 33(13). 1455-1461 (2003)

Kawazu S、Nozaki T、Tsuboi T 等人：“啮齿动物疟疾寄生虫约氏疟原虫的过氧化还原蛋白的表达谱。”国际寄生虫学杂志。

Katakura K, Fujise H, Takeda K, et al.: "Overexpression of LaMDR2, a novel multidrug resistance ATP-binding cassette transporter, causes 5-fluorouracil resistance in Leishmania amazonensis."FEBS Letters. 561(1-3). 207-212 (2004)

Katakura K、Fujise H、Takeda K 等人：“LaMDR2（一种新型多药耐药性 ATP 结合盒转运蛋白）的过度表达会导致亚马逊利什曼原虫产生 5-氟尿嘧啶耐药性。”FEBS Letters。

Ling IT, Florens L, Dluzewski AR, Kaneko O, et al.: "The Plasmodium falciparum clag9 gene encodes a rhoptry protein that is transferred to the host erythrocyte upon invasion."Molecular Microbiology. (in press). (2004)

Ling IT、Florens L、Dluzewski AR、Kaneko O 等人：“恶性疟原虫 clag9 基因编码一种棒状体蛋白，该蛋白在入侵时转移至宿主红细胞。”分子微生物学。

Arakawa T, Tsuboi T, Kishimoto A, Sattabongkot J, Suwanabun N, Rungruang T, Matsumoto Y, Tsuji N, Hisaeda H, Stowers A, Shimabukuro I, Sato Y, Torn M: "Serum antibodies induced by intranasal immunization of mice with Plasmodium vivax Pvs25 co-administered

Arakawa T、Tsuboi T、Kishimoto A、Sattabongkot J、Suwanabun N、Rungruang T、Matsumoto Y、Tsuji N、Hisaeda H、Stowers A、Shimabukuro I、Sato Y、Torn M：“用疟原虫鼻内免疫小鼠诱导的血清抗体

Tsuboi T, Kaneko O, Eitoku C, Suwanabun N, Sattabongkot J, Vinetz JM, Torn M.: "Gene structure and ookinete expression of the chitinase genes of Plasmodium vivax and Plasmodium yoelii"Molecular and Biochemical Parasitology. 130(1). 51-54 (2003)

Tsuboi T、Kaneko O、Eitoku C、Suwanabun N、Sattabongkot J、Vinetz JM、Torn M.：“间日疟原虫和约氏疟原虫几丁质酶基因的基因结构和动合子表达”分子和生化寄生虫学。