Molecular cloning of a Babesia caballi gene encoding the 134-kilodalton protein and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay

Abstract

A Babesia caballi gene encoding the 134-kDa (BC134) protein was immunoscreened with B. caballi-infected horse serum. An enzyme-linked immunosorbent assay (ELISA) using recombinant BC134 protein could effectively differentiate B. caballi-infected horse sera from Babesia equi-infected or noninfected control horse sera. These results suggest that the recombinant BC134 protein is a potential diagnostic antigen in the detection of B. caballi infection.

FIG. 1.

Complete nucleotide sequence, including the 5′ and 3′ untranslated regions, of the BC134 gene. The amino acid sequence translated from the long ORF is depicted under the nucleotide sequence. The underlining shows a predicted signal peptide sequence.

FIG. 2.

Purification of recombinant GST-BC134 protein with glutathione-Sepharose 4B beads. Lanes: a, whole lysate of the transformed E. coli with pGEX/BC134; b, pellet fraction from the whole lysate; c, supernatant fraction from the whole lysate; d and e, purified GST-BC134 and GST proteins, respectively.

FIG. 3.

Western blot analysis of the lysates of B. caballi (lane a)- and B. equi (lane b)-infected and noninfected (lane c) equine erythrocytes incubated with mouse immune serum against recombinant BC134 protein. The positions of the standard molecular masses (in kilodaltons) are indicated on the left side of the panels. Note that 134 kDa of the native protein is seen only in the lysate of B. caballi-infected erythrocytes.

FIG. 4.

Localization of BC134 protein as shown by ethanol-acetone-fixed smears of B. caballi-infected erythrocytes incubated with the anti-recombinant BC134 protein mouse immune serum and then observed by confocal laser scanning microscopy. The immune reaction (green) and nucleus (red) were visualized with the fluorescein isothiocyanate-conjugated secondary antibody and propidium iodide staining, respectively. Bars, 5 μm.

FIG. 5.

ELISA showing the reactivity of the GST-BC134 protein to horse sera. Dots represent OD₄₁₅ of B. caballi-infected (lane 1), B. equi-infected (lane 2), and noninfected (lane 3) horse sera.