Immunogenicity and antigenicity of the recombinant EMA-1 protein of Theileria equi expressed in the yeast Pichia pastoris

Abstract

The equine piroplasmosis caused by Theileria equi is one of the most important parasitic diseases of the equine, causing damage to animal health and economic losses. In T. equi, 2 merozoite surface proteins, equi merozoite antigen EMA-1 and EMA-2, have been identified as the most immunodominant antigens. This suggests that these antigens might be used as immunobiological tools. The EMA-1 of Theileria equi was cloned and expressed in the yeast Pichia pastoris. The transformed yeast was grown at high cell density, expressing up to 389 mg.L -1 of recombinant protein. The protein was concentrated and detected in Dot blot. The recombinant product was antigenically similar to the native protein as determined using monoclonal antibodies, and polyclonal antibodies obtained from equines naturally infected with T. equi. The immunogenicity of rEMA-1 protein was demonstrated by IFAT using sera from recombinantprotein-immunized mice using aluminum hydroxide as adjuvant. All animals vaccinated with rEMA-1 developed a high specific antibody response. This results suggest that rEMA-1expressed in P. pastoris might be a strong candidate to be used as an antigen for immune diagnostics as well as a vaccine antigen.Keywords: Theileria equi, Pichia pastoris, EMA-1, recombinant vaccine. ResumoA piroplasmose equina causada por Theileria equi é uma das mais importantes doenças parasitárias de equídeos, causando danos a saúde animal e perdas econômicas. Em T. equi, 2 proteínas de superfície de merozoítos, equi merozoite antigen EMA-1 e EMA-2, têm sido identificadas como antígenos imunodominantes. Sugerindo que estes antígenos possam ser usados como produtos imunobiológicos. O gene EMA-1 de T. equi foi clonado e expressado na levedura Pichia pastoris. As leveduras transformadas foram cultivadas a altas densidades celulares expressando 389 mg.L -1 de proteína recombinante. A proteína foi concentrada e detectada em Dot blot. O produto recombinante foi antigenicamente similar à proteína nativa quando determinado usando anticorpo monoclonal e anticorpos policlonais obtidos de equinos naturalmente infectados com T. equi. A imunogenicidade da proteína rEMA-1 foi demonstrada por RIFI utilizando soro de camundongos imunizados com proteína recombinante usando hidróxido de alumínio como adjuvante. Todos os animais vacinados com rEMA-1 desenvolveram uma alta resposta específica de anticorpos. Esses resultados sugerem que rEMA-1 expressa em P. pastoris possa ser um candidato para ser usado como antígeno para diagnóstico imunológico bem como antígeno para vacinas.Palavras-chave: Theileria equi, Pichia pastoris, EMA-1, vacina recombinante.

Expression cassette and plasmid construction for Yeast Surface Display in Saccharomyces cerevisiae

“…Washing step was repeated and membranes were placed in the chromogenic substrate solution, containing 6 µg of DAB (3,3'-diaminobenzidine), 50 mM Tris HCl, 0.3% nickel sulfate solution and 10 µL of hydrogen peroxide (H 2 O 2 ). Biomass samples from the control culture BY4741 pYES2, BY4741 pYES2/gDαAgglutinin and their supernatants were tested against recognition by anti-histidine mAb, following the adaptation of the dot blot protocol published at Nizoli et al (2009). Indirect immuno uorescence assay Samples of BY4741 pYES2 and BY4741 pYES2/gDαAgglutinin cultures, both at a concentration of 10 7 cells/mL, were xed (30 min at 30 °C) over indirect immuno uorescence slides.…”

“…The immunogenic potential of glycoprotein D (gD) of bovine herpesvirus type 5 (BoHV-5) has been studied by our research group for some years, being the protein expressed recombinantly (rgD) using Escherichia coli and Pichia pastoris expression systems. In previous studies, our group have showed that rgD used as a vaccinal antigen was able to induce high neutralizing antibodies titers in mice and cattle (Nizoli et al 2009;Araujo et al 2018), and an ELISA test (Enzyme-Linked Immunosorbent Assay) composed of rgD proved the protein recognition by the serum of naturally infected animals (Dummer et al 2016). Based on the extensive knowledge we achieved using gD, it became a model protein for different studies in our laboratory.…”

In Silico Screening of Putative Corynebacterium pseudotuberculosis Antigens and Serological Diagnosis for Caseous Lymphadenitis in Sheep by Enzyme-Linked Immunosorbent Assay

“…This suggests the recombinant protein preserves the antigenic potential, even after heterology expression in E. coli . The Dot Blot is a technique widely used to determine the antigenic property of recombinant protein [ 43 ].…”

Oxidative Damage Inflicted by Theileria equi on Horse Erythrocytes When Cultured In Vitro by Microaerophilous Stationary Phase Technique

“…The infection caused by T. equi parasite is more widespread and pathogenic. The animal infected with T. equi parasite showed decrease in the performance activity might be due to less tissue oxygenation caused by infecting parasite leading to destruction of red blood cells (RBCs) [1].…”