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Journal of Clinical Microbiology, July 2001, p. 2603-2609, Vol. 39, No. 7
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.7.2603-2609.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Identification and Expression of a 50-Kilodalton Surface Antigen of Babesia gibsoni and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

Shinya Fukumoto, Xuenan Xuan, Yoshifumi Nishikawa, Noboru Inoue, Ikuo Igarashi, Hideyuki Nagasawa, Kozo Fujisaki, and Takeshi Mikami*

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080 to 8555, Japan

Received 8 January 2001/Returned for modification 20 March 2001/Accepted 8 April 2001

A cDNA expression library prepared from Babesia gibsoni merozoite mRNA was screened with B. gibsoni-infected dog serum. cDNA encoding a 50-kDa protein was cloned and designated the P50 gene. The complete nucleotide sequence of the P50 gene was 1,922 bp. Computer analysis suggested that the sequence of the P50 gene contained an open reading frame of 1,401 bp with a coding capacity of approximately 50 kDa. The complete genomic nucleotide sequence of the P50 gene has been analyzed and shown to contain a single intron of 37 bp. Southern blotting analysis indicated that the P50 gene was present at a single copy in the B. gibsoni genome. The native P50 protein of B. gibsoni with a molecular mass of 50 kDa was identified by Western blotting with anti-recombinant P50 mouse serum. Confocal laser microscopic analysis showed that the P50 protein was located on the surface of B. gibsoni merozoites. The recombinant P50 protein expressed by baculovirus in insect cells was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Furthermore, the antibody response against the recombinant P50 protein was maintained until the chronic stage of infection in dogs experimentally infected with B. gibsoni was developed. These results demonstrate that the recombinant P50 protein might be a useful diagnostic reagent for detection of antibodies to B. gibsoni in dogs.

* Corresponding author. Mailing address: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. Phone: 81-155-49-5648. Fax: 81-155-49-5643. E-mail: gen{at}obihiro.ac.jp.

Journal of Clinical Microbiology, July 2001, p. 2603-2609, Vol. 39, No. 7
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.7.2603-2609.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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