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

Document Type : Original Articles


1 Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran

2 Department of Parasitology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran

3 Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran

4 Department of Clinical Sciences, Faculty of Veterinary Medicine, Shiraz University, Shiraz, Iran


Diversity among the pathogenic strains of Theileria equi (T. equi), 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 (EMA-1 gene), as one of the most important immunodominant surface proteins in T. equi, from naturally infected horses in Iran. The immunogenic region of EMA-1 gene was amplified using the blood of infected horses. EMA-1 gene was cloned into pET26b vector. Then, recombinant plasmids (pET 26b-EMA-1) were transformed into competent E. coli 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 EMA-1 gene. Sequence analysis revealed that cloned EMA-1 and protein had 94% and 97% homology to EMA-1 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 EMA-1 protein with a 28-kDa band. The results of the dot blot analysis revealed that the horse serum containing antibody against T. equi could react with the purified recombinant protein. Purified EMA-1 protein can be used as a reliable tool for the future development of diagnostic tests or vaccines.


Main Subjects

Article Title [French]

Le Clonage et l'Expression de Régions Immunogènes du Gène EMA-1 du Parasite de Theileria equi Isolé de Chevaux Infectés

Abstract [French]

La variation parmi les souches immunogènes de Theileria equi, la principale cause de la piroplasmose chez les chevaux, peut affecter la reconnaissance du parasite et de l'immunogénicité de l'hôte. La production de protéines recombinantes à partir de parasites des chevaux infectés dans les zones endémiques fournit un outil d'identification de l'immunité parasitaire. Le but de la présente étude est le clonage, l'expression et la purification de régions immunogènes de la protéine EMA-1 (l'une des plus importantes protéines de surface immunogènes dans Theileria equi) chez les chevaux infectés. Les régions immunogènes de la protéine EMA-1 du sang des chevaux infectés ont été reproduites. Le gène EMA-1 a été cloné dans un vecteur pET 26b. Ensuite, le plasmide recombinant (pET 26b-EMA-1) a été transféré dans le récepteur E. coli BL21. La confirmation du clonage a été réalisée par PCR, découpe de vecteurs avec des enzymes de découpe et analyse du séquençage de l’ADN. L'expression de la protéine recombinante a été induite en utilisant de l'IPTG. Une purification a été réalisée en utilisant des colonnes NI-NTA et en vérifiant l'expression en utilisant une SDS-PAGE à 10%, et le Dot blot en utilisant des anticorps anti-HisTag. La réponse immunitaire de la protéine recombinante avec le sérum du cheval infecté a été évaluée en utilisant le test de Dot Blot. L'analyse du produit de PCR a montré une bande de750 pb appartenant au gène EMA-1. L'analyse de séquence du gène et de la protéine EMA-1 avec d'autres séquences de la banque de gènes a montré une similarité de 94 et 97%, respectivement. L'analyse de séquence a confirmé la coupe avec des enzymes pour une insertion correcte du vecteur dans le vecteur. L'analyse SDS-PAGE a montré l'expression de la protéine EMA-1 avec une bande de protéine de 28 kDa. L'analyse des résultats de Dot Blot a montré que le sérum du cheval contenant des anticorps contre Theileria equi pouvait réagir avec la protéine recombinante purifiée. La protéine purifiée d'EMA-1 peut être utilisée comme un outil fiable pour la conception de tests de diagnostic et de tests de vaccins à l'avenir.

Keywords [French]

  • Theileria equi
  • Gène EMA-1
  • Clonage
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