Molecular characterization of a novel 32-kDa merozoite antigen ofBabesia gibsoniwith a better diagnostic performance by enzyme-linked immunosorbent assay

Abstract

SUMMARYWe cloned and expressed a novel gene encoding a 32-kDa merozoite protein ofBabesia gibsoni(BgP32). The length of nucleotide sequence of the cDNA was 1464 bp with an open reading frame of 969 bp. The truncated recombinant BgP32 (rBgP32) without a signal peptide and C-terminal hydrophobic sequence was expressed inEscherichia colias a soluble glutathione-S-transferase (GST) fusion protein. Western blotting demonstrated that the native protein was 32-kDa, consistent with molecular weight of the predicted mature polypeptide. Enzyme-linked immunosorbent assay (ELISA) using rBgP32 detected specific antibodies from 8 days to 541 days post-infection in the sequential sera from a dog experimentally infected withB. gibsoni. Moreover, the antigen did not cross-react withB. canissubspecies and closely related protozoan parasites, indicating that rBgP32 is a specific diagnostic antigen. Analysis of 47 sera taken from dogs with anaemic signs revealed that rBgP32 detected a higher proportion ofB. gibsoniseropositive samples (77%) than its previously identified rBgP50 (68%) homologue. These results indicate that the BgP32 is a novel immunodominant antigen ofB. gibsoni, and rBgP32 might be useful for diagnosis ofB. gibsoniinfection.