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Brucellosis is one of the most common diseases in humans and it has a worldwide spread. The design and production of newly synthesized proteins can be served as a goal for the rapid and accurate detection of brucellosis. To this aim, finding the antigenic epitopes is the first step to design a diagnostic method. In this study, the epitope mapping procedure was carried out by IEDB analysis resource using Flagellin and Porin amino acid sequences. The selected sequences were linked by GS linkers and cloned into pET26b vector. After confirmation of the expressed recombinant polypeptide by western blotting, it was immunologically analyzed by gel diffusion assay. The SDS PAGE and western blot analysis confirmed the 27 KDa polypeptide production and observing an arc in gel diffusion test demonstrated the precipitation of serum antibodies and the presence of specific antigen complexes. The results showed that the recombinant polypeptide produced in E. coli BL21, could interact with antibodies present in Brucella immunized sheep serum. HIGHLIGHTS •Prediction of antigenic epitopes for Brucella membrane proteins. •Production of designed polypeptide in E. coli BL21 (DE3). •Interaction of produced polypeptide with Brucella specific antibodies.