| Popis: |
Ookinete surface proteins are known to be successful targets for transmission blocking vaccines (TBVs) that can achieve significant reductions in Plasmodium transmission. To date, only three proteins have been definitively localised to the ookinete surface; P25, P28 and CTRP. To identify further proteins, this study produced an ookinete surface-enriched proteome (OSEP) of P. berghei; Cultured and purified ookinetes were surface-biotinylated. Labelled proteins were then affinity-purified and subjected to high-throughput mass spectrometry (MudPIT). The resulting proteome shows significant enrichment of known and predicted surface proteins. However, it also contains known intracellular proteins, due to ookinete permeabilisation during labelling. Twelve candidate surface proteins were selected from the generated proteome based on their significant enrichment in the OSEP and the bioinformatic prediction of features associated with surface proteins. To produce antibodies, DNA immunisation was performed using plasmids encoding candidate proteins. Antibodies were successfully generated against three proteins, one recognising an ookinete-specific protein and a second recognising a protein of the asexual stages, gametocytes and ookinetes. Immunofluorescence failed to localise candidate proteins to either the ookinete or to other parasite stages. Ookinete-specific expression seen in Western blots, promoted further work on candidate protein 0250. However, a knockout of this gene failed to result in a phenotype distinct from that of wild-type parasites and eGFP-tagging failed to localise this protein. The generated OSEP provides a source of potential ookinete surface proteins. It is hoped that investigation these proteins will lead to novel targets for TBV and to a deeper understanding of how the ookinete interacts on a molecular level with its environment |
