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Journal of Clinical Microbiology, November 1999, p. 3475-3480, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cloning and Expression of a 48-Kilodalton Babesia caballi Merozoite Rhoptry Protein and Potential Use of the Recombinant Antigen in an Enzyme-Linked Immunosorbent Assay

Hiromi Ikadai,1 Xuenan Xuan,1 Ikuo Igarashi,1,* Shigeyasu Tanaka,2 Takumi Kanemaru,3 Hideyuki Nagasawa,1 Kozo Fujisaki,1 Naoyoshi Suzuki,1 and Takeshi Mikami1

The Research Center for Protozoan Molecular Immunology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555,1 Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529,2 and Epizootic Research Station, Equine Research Institute, The Japan Racing Association, Tochigi 329-0412,3 Japan

Received 21 April 1999/Returned for modification 4 June 1999/Accepted 21 July 1999

A cDNA expression library prepared from Babesia caballi merozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B. caballi merozoite. A cDNA encoding a 48-kDa protein of B. caballi was cloned and designated BC48. The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron. Southern blotting analysis indicated that the BC48 gene contained more than two copies in the B. caballi genome. Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa. The recombinant protein expressed by the vaccinia virus vector in horse cells had an apparent molecular mass of 48 kDa, which was the same as that of the native B. caballi 48-kDa protein. Moreover, recombinant proteins expressed by the pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion proteins were used for antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate very clearly between B. caballi-infected horse sera and B. equi-infected horse sera or noninfected normal horse sera. These results suggest that this simple and highly sensitive test might be applicable to the detection of B. caballi-infected horses in the field.

* Corresponding author. Mailing address: The Research Center for Protozoan Molecular Immunology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan. Phone: 81-155-49-5641. Fax: 81-155-49-5643. E-mail: igarcpmi{at}obihiro.ac.jp.

Journal of Clinical Microbiology, November 1999, p. 3475-3480, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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