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Molecular characterizations of three distinct Babesia gibsoni rhoptry-associated protein-1s (RAP-1s)

Published online by Cambridge University Press:  27 July 2009

M. A. TERKAWI ,
A. AMORNTHEP ,
H. OOKA ,
G. ABOGE ,
H. JIA ,
Y.-K. GOO ,
B. NELSON ,
J. YAMAGISHI ,
Y. NISHIKAWA  and
I. IGARASHI
...Show all authors
M. A. TERKAWI
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
A. AMORNTHEP
Affiliation:
Department of Veterinary Public Health and Diagnostic Service, Faculty of Veterinary Medicine, Kasetsart University, Kamphaengsan Campus, Nakhonpathom 73140, Thailand
H. OOKA
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
G. ABOGE
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
H. JIA
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
Y.-K. GOO
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
B. NELSON
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
J. YAMAGISHI
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
Y. NISHIKAWA
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
I. IGARASHI
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
S.-I. KAWAZU
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
K. FUJISAKI
Affiliation:
Department of Frontier Veterinary Medicine, Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan
X. XUAN*
Affiliation:
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
*
*Corresponding author: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. Tel: +81 155 49 5648. Fax: +81 155 49 5643. E-mail: gen@obihiro.ac.jp
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Summary

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.

Keywords

Babesia gibsonirhoptry-associated proteinantigenicity
Type
Research Article
Information
Parasitology , Volume 136 , Issue 10 , September 2009 , pp. 1147 - 1160
Copyright
Copyright © Cambridge University Press 2009

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