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

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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.