Gene cloning of a novel merozoite rhoptry protein from Plasmodium falciparum

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By the peptide sequencing from the bands obtained from the immuno-affinity purified 140 kDa rhoptry protein of Plasmodium yoelii, we obtained partial peptide sequences of PyRhopH1. To determine the gene encoding P.yoelii 140-kDa rhoptry protein, PCR amplifications were done from cDNA using degenerated oligonucleotides designed based on the partial amino acid sequences. Finally, we have identified the Plasmodium RhopH1 in P.yoelii.By the PCR-based gene-walking, we completed gDNA and cDNA sequences that encoded an open reading frame for 1292 amino acids protein separated by 7 introns. Deduded amino acid sequence has putative secretory signal sequence at the N-terminus (aa 1-24) predicted by SignalP and no apparent transmembrane domain. TBLASTN analysis against P.yoelii genome database revealed the existence of a paralogue of this gene, thus we designated the gene identified from peptide sequences as pyrhophla and second gene found in the database as pyrhoph1b. Using deduced amino acid sequence of pyrhophla as a query, TBLASTN search against P.falciparum genome database identified a gene family coding PyRhopH1 orthologues. To confirm the size and localization of PyRhopH1A, Western immunoblot analysis and immunofluorescent microscopy were performed. Antiserum generated with pYRH1A DNA vaccine reacted only 140-kDa band on the Western immunobloting, and showed a punctate pattern typical for the apical organelle as same as the pattern by anti-PyRhopH1 mAb by immunofluorescent microscopy.