High-level expression and purification of a truncated merozoite antigen-2 of Babesia equi in Escherichia coli and its potential for immunodiagnosis.
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.
Huang
Xiaohong
X
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
Xuan
Xuenan
X
Yokoyama
Naoaki
N
Xu
Longshan
L
Suzuki
Hiroshi
H
Sugimoto
Chihiro
C
Nagasawa
Hideyuki
H
Fujisaki
Kozo
K
Igarashi
Ikuo
I
D000818:Animals; D000918:Antibody Specificity; D000953:Antigens, Protozoan / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001403:Babesia / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D001404:Babesiosis / Q000175:diagnosis / Q000276:immunology; D015153:Blotting, Western; D002681:China; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D006736:Horses; D007159:Immunologic Tests; D007700:Kinetics; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D000953:Antigens, Protozoan; C111468:EMA-2 protein, Babesia equi; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011994:Recombinant Proteins