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

侵袭型疟疾寄生虫利用其顶端细胞器，如棒状体，进行红细胞入侵。其中一种吸管分子，RHopH复合体，已知与红细胞表面结合。为了阐明红细胞膜受体的特征和Rhop H复合体的结合域，我们承诺今年将研制一组针对恶性疟原虫Rhop H复合体的重组蛋白和抗体。首先，我们对表达载体进行了修饰，添加了myc-tag和绿色荧光蛋白，以便于下游分析。其次，我们采用了Gateway system(Invitgen)来使表达构建变得更容易，因为如果没有这种技术，很难制作最终的大质粒构建。第三，我们将基因分析认为是RHopH复合体中结合成分最强的候选基因Rhop H1分成3个部分，并连接到表达载体中。目前，我们正在评估这些构建物在哺乳动物系统中的表达情况。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Rhop H复合体各组分的特异性抗体。通过DNA免疫获得了特异的抗血清。免疫荧光显微镜和免疫印迹分析表明，这些血清对寄生虫天然蛋白有反应。目前，这些都被用于免疫沉淀RHopH复合体，该复合体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。

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.