Molecular cloning and characterization of Babesia orientalis rhoptry-associated protein 1

- Babesia orientalis rhoptry-associated protein 1 (BoRAP-1) gene was determined, and this is the first report of BoRAP-1.
- The full length of BoRAP-1 gene is 1732 bp with an open reading frame of 1434 bp, encoding 478 amino acid residues with a predicted size of 52.5 kDa.
- The native BoRAP-1 existed on B. orientalis merozoites.
- BoRAP-1 could be a diagnostic antigen for detecting B. orientalis infection.

The rhoptry-associated protein 1 (RAP-1) gene of Babesia orientalis was obtained from a cDNA expression library by immunoscreening with B. orientalis-infected water buffalo sera. The nucleotide sequence of the cDNA was 1732 bp with an open reading frame (ORF) of 1434 bp, encoding a polypeptide of 478 amino acid residues with a predicted size of 52.5 kDa. The ORF was cloned into a pGEX-KG plasmid and subsequently expressed as a GST-fusion protein. The recombinant RAP-1 of B. orientalis (rBoRAP-1) was purified and evaluated as an antigen using Western blotting. The native BoRAP-1 was recognized by the antibodies raised in rabbits against rBoRAP-1. Strong immunofluorescence signals were observed in erythrocytes infected with B. orientalis. Phylogentic analysis revealed that B. orientalis fell into a Babesia clade and most closely related to Babesia bovis and Babesia ovis, which was similar to the previous reported trees based on 18S rRNA and HSP70 genes. The present study suggests that the BoRAP-1 might be a potential diagnostic antigen, and the RAP-1 genes can aid in the classification of Babesia and Theileria species.