A unique Babesia bovis spherical body protein is conserved among geographic isolates and localizes to the infected erythrocyte membrane

Using monoclonal antibody (mAb) 70/52.9, generated from a Babesia bovis is fraction enriched for spherical body organelles, we have identified a 135-k Da protein containing an epitope conserved in B. bovis strains from Texas, Mexico, and Australia. The protein was localized to the spherical bodies of the babesial apical complex and was designated spherical body protein 3 (S BP3), according to the established nomenclature. Immunofluorescence studies showed binding of the 70/52.9 mAb to the infected-erythrocyte membrane reg ion but not to their uninfected counterparts, demonstrating a host-cell ass ociation shared with the previously isolated B. bovis spherical body protei ns, SBP1 and SBP2. Using mAb 70/52.9, the full-length cDNA encoding SBP3 wa s isolated from an expression library, sequenced, and oligonucleotide prime rs synthesized to amplify the genomic copy by polymerase chain reaction. Th e genomic copy contained no introns and was identical to the cDNA sequence with each containing a single, large open reading frame encoding a protein of 1089 residues. Analysis of the SBP3 amino acid sequence revealed no sign ificant amino acid identity to SBP1 and SBP2 and a lack of repeated epitope s, a notable feature of the other two spherical body proteins. Labeled prob es derived from the coding region of SBP3 hybridized to single fragments on Southern blots containing B. bovis genomic DNA indicating a single copy ge ne. With the identification of this third spherical body protein, which ass ociates with the cytoplasmic face of the infected-erythrocyte membrane, a c omplement of distinct B. bovis proteins have been identified that are likel y to contribute to intracellular survival, growth, and development for this parasite. The encoded protein should be valuable for functional investigat ions and evaluation of potential targets for host immunity. (C) 2000 Publis hed by Elsevier Science B.V. All rights reserved.