الفهرس 
Materials and methodsOnly 14 pages are availabe for public view
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Abstract
Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone consists of an ORF of 1,995 bp encoding 664 amino acid residues encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. The translated BgGARP contained a glutamic acid-rich region profile at amino acid residues (174-583 aa) in the C-terminal, therefore, the protein was designated as Babesia gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of the translated
mice (Clea, Japan) were intraperitoneally immunized with 100 μg of purified rBgGARPt emulsified with an equal volume of complete Freund’s adjuvant (Difco Laboratories, USA). Two additional boosters consisting of 50 μg of rBgGARPt emulsified with incomplete Freund’s adjuvant (Difco Laboratories, USA)
were intraperitoneally administered at 2 weeks intervals. The mice were bled 16 days after the last booster, and serum samples were stored at -30 ◦C. Mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs. In conclusion, a novel gene encoding 76-kDa proteins from the B. gibsoni cDNA expression library was characterized, and its capacity as diagnostic antigen was evaluated. We demonstrated that ELISA based on rBgGARPt has enough specificity and sensitivity for performing epidemiological surveys.