High-level expression and purification of a truncated merozoite antigen-2 of Babesia equi in Escherichia coli and its potential for immunodiagnosis

Abstract

The gene encoding a truncated merozoite antigen-2 (EMA-2t) of Babesia equi was cloned and highly expressed in Escherichia coli as a glutathione S-transferase fusion protein (G-rEMA-2t). Both G-rEMA-2t and rEMA-2t (after the removal of glutathione S-transferase) had good antigenicity. Either Western blot analysis with rEMA-2t or enzyme-linked immunosorbent assay (ELISA) with G-rEMA-2t clearly discriminated the sera of horses experimentally infected with B. equi from sera of horses infected with Babesia caballi and healthy horses, although rEMA-2t was not suitable for ELISA, probably owing to its poor absorbability to the plates. The specific antibodies in B. equi-infected horses were detectable during both acute and latent infection (6 to 244 days postinfection). Horse sera from Jilin Province, China, were examined by the two tests. The seroprevalence of B. equi was 49.2% (31 of 63 sera) by Western blot analysis with rEMA-2t and 47.6% (30 of 63 sera) by ELISA with G-rEMA-2t. The correspondence was 98.4% (62 of 63 sera) between the two tests. The results indicate that G-rEMA-2t and rEMA-2t proteins should be suitable antigens for the development of an effective immunodiagnostic assay due to their high sensitivity, specificity, and great yield.

FIG. 1.

Hydrophilicity plot of EMA-2 antigen sequence and location of EMA-2t. The plot shown was derived from the amino acid sequence of the open reading frame of the EMA-2 gene by using a computer analysis programs (window = 7) developed by Hopp and Woods (7). nt, nucleotide.

FIG. 2.

Expression of the recombinant fusion protein of entire (lanes 1 and 2) and truncated (lanes 3 and 4) EMA-2 in E. coli. Lane M, molecular mass standard; lanes 1 and 3, proteins in soluble fractions; lanes 2 and 4, proteins in insoluble fractions. Proteins were stained with amido black 10B.

FIG. 3.

Purified G-rEMA-2t and rEMA-2t and their antigenicity. (A) Antigens stained with amido black 10B; (B) reactivity of G-rEMA-2t and rEMA-2t to serum from B. equi-infected horse by Western blot analysis. Lane M, molecular mass standard; lane 1, G-rEMA-2t (51 kDa); lane 2, rEMA-2t (25 kDa); lane 3, GST; lane 4, B. equi-infected erythrocyte lysate; lane 5, normal erythrocyte lysate.

FIG. 4.

ELISA with G-rEMA-2t. Group 1, sera from normal horses; groups 2 and 3, sera from horses infected experimentally with B. equi and B. caballi, respectively; group 4, sera from horses in Jilin Province, China.

FIG. 5.

Comparison of results obtained by CFT (A), ELISA (B), Western blot analysis (C), and microscopic examination (D) in the diagnosis of B. equi infection. E3 and E4 were two horses experimentally infected with B. equi. C4 and C5 were two horses experimentally infected with B. caballi.