title: 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.
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Huang
authors.forename: Xuenan
authors.initials: X
authors.lastname: Xuan
authors.forename: Naoaki
authors.initials: N
authors.lastname: Yokoyama
authors.forename: Longshan
authors.initials: L
authors.lastname: Xu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Suzuki
authors.forename: Chihiro
authors.initials: C
authors.lastname: Sugimoto
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Nagasawa
authors.forename: Kozo
authors.initials: K
authors.lastname: Fujisaki
authors.forename: Ikuo
authors.initials: I
authors.lastname: Igarashi
mesh_terms: 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
chemical_list: D000953:Antigens, Protozoan; C111468:EMA-2 protein, Babesia equi; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011994:Recombinant Proteins