The aim of the present study is to provide a strategy for predicting the production of antigenic regions of Clostridium novyi alpha toxin. The selection is based on B-cell epitopes and MHCII binding protein by immunoinformatics tools. The study resulted in identifying antigenic regions in the beginning and middle (3–17 and 965–997 amino acid residues) as suitable binders to MHCII and the carboxyl terminal of the protein (1800–1958 amino acid residues) in B-cell epitopes. The appropriate region in B-cell epitopes was chosen for cloning. The presence of recombinant protein was detected with immunological methods. Subsequently, BALB/C mice were immunized with the recombinant protein and alpha toxin, and the antibodies produced were evaluated by dot-blot and ELISA tests. After cloning the highly antigenic region (1799–1966 amino acid residues), the results of immunological tests showed that the recombinant protein reacts with antitoxin antibodies. Antirecombinant protein has a higher affinity to the alpha toxin than antialpha toxin. Thus, the carboxyl terminal of the protein (1799–1966 amino acid residues) in B-cell epitopes could be a proper candidate for a peptide vaccine against alpha toxin.

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