Cloning and Expression of Immunogenic Regions of EMA-1 Gene of Theileria equi From Infected Horses

Abstract

Diversity among the pathogenic strains of<em> Theileria equi</em> (<em>T. equi</em>), a major agent of equine piroplasmosis, can affect the appropriate detection of parasite and host immunization. Production of recombinant surface proteins from an infected horse in natural endemic area provides a reliable tool for immunodiagnosis of parasite. Regarding this, the present study was targeted toward the cloning, expression, and purification of the immunogenic regions of equine merozoite antigen 1 (<em>EMA-1 </em>gene), as one of the most important immunodominant surface proteins in <em>T. equi</em>, from naturally infected horses in Iran. The immunogenic region of <em>EMA-1</em> gene was amplified using the blood of infected horses. <em>EMA-1</em> gene was cloned into pET26b vector. Then, recombinant plasmids (pET 26b-EMA-1) were transformed into competent <em>E. coli </em>BL21 (DE3) cells. Cloning was confirmed by polymerase chain reaction (PCR), restriction enzyme assays, and DNA sequence analysis. The recombinant protein was expressed using isopropyl β-D-1-thiogalactopyranoside as an inducer, purified using nickle-nitrilotriacetic acid column, and then confirmed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and dot blot analysis utilizing Anti-His Tag antibody. Furthermore, the immunoreactivity of recombinant protein against the serum of the infected horses was evaluated using dot blot analysis. The PCR product analysis showed a 750-bp band belonging to immunogenic regions of <em>EMA-1</em> gene. Sequence analysis revealed that cloned <em>EMA-1 </em>and protein had 94% and 97% homology to <em>EMA-1</em> sequences submitted to GenBank from different countries, respectively. Restriction enzyme and sequence analyses confirmed the subcloning and correction of the orientation of inserted gene. The SDS-PAGE analysis confirmed the expression of <em>EMA-1</em> protein with a 28-kDa band. The results of the dot blot analysis revealed that the horse serum containing antibody against <em>T. equi</em> could react with the purified recombinant protein. Purified <em>EMA-1</em> protein can be used as a reliable tool for the future development of diagnostic tests or vaccines.