1. Molecular characterizations of three distinct Babesia gibsoni rhoptry-associated protein-1s (RAP-1s).
- Author
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Terkawi, M. A., Amornthep, A., Ooka, H., Aboge, G., Jia, H., Goo, Y.-K., Nelson, B., Yamagishi, J., Nishikawa, Y., Igarashi, I., Kawazu, S.-I., Fujisaki, K., and Xuan, X.
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RECOMBINANT proteins ,PERFORMANCE-enhancing drugs ,ENZYME-linked immunosorbent assay ,RECOMBINANT molecules ,IMMUNOLOGY technique ,IMMUNOFLUORESCENCE - Abstract
Three cDNAs encoding rhoptry-associated protein 1 (RAP-1) homologues were found in the Babesia gibsoni EST database. Based on similarities to BgRAP-1a, which was identified previously by serological screening of a cDNA merozoite library, the two new genes were designated BgRAP-1b (33.7%) and BgRAP-1c (57%). Mice antiserum raised against each recombinant protein reacted specifically with B. gibsoni parasites as determined by Western blotting, which showed native molecular sizes of the BgRAP-1a (51 kDa), BgRAP-1b (53 kDa) and BgRAP-1c (47 kDa) consistent with predictable molecular weights. Immunofluoresence using these antibodies revealed localization of all BgRAP-1s within the matrix of merozoites; however, BgRAP-1a appeared to diverge from the other two when it was found secreted into the cytoplasm of infected erythrocytes. Apical localization of all 3 BgRAP-1s during the extracellular stage of the parasite combined with their ability to bind a canine erythrocyte membrane fraction was suggestive of a role for these proteins in erythrocyte attachment. Lastly, the ability of these recombinant proteins to be used as diagnostic reagents was tested by ELISA and the sensitivities of BgRAP-1a and BgRAP-1c were found increased through N-terminal truncation. Taken together, our data suggest divergent roles for the 3 BgRAP-1s in the merozoite stage of B. gibsoni. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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