Construction of chimeric protein 3M2e.FliC and its immunoinformatics analyses and expression in Bacillus subtilis

Introduction: Influenza A virus causes unpredictable epidemics and pandemics by creating antigenic variations. With the appearance of each new strain, rapid emergency countermeasures are taken against this new strain. Hence, designing an applicable and cross protective strategy to counter this virus is of great importance. To achieve this, choosing conserved antigenic regions in influenza virus proteins for making a universal vaccine is one of the best options. M2 channel in influenza virus membrane has a conserved sequence in ectodomain region called M2e. This region is the same between the majority of influenza A virus strains. But this peptide region is not a good stimulant for the immune system due to its short length. Immunoadjuvant property of Salmonella Typhimurium flagellin (FliC) has been previously shown to arouse immunity in the mucosal region. A combination of M2e and FliC was used in a construct in this study. Methods: To increase the possibility of immunugencity of this construct, in silico predictions of this fusion peptide construct were performed first. Three repeats of M2e gene and FliC sequence were cloned into pET28 vector and then were sub-cloned to pHT43. Finally, the construct was transformed into Bacillus subtilis by electroporation. After IPTG induction, the total cell protein and the supernatant protein were analyzed via protein analyses methods. Results: Based on in silico immunogenicity results, the designed recombinant peptide was deemed suitable to be used as a universal influenza vaccine candidate. The validity of pHT43.3M2e.FliC construct was confirmed by restriction map analysis. Western-blotting confirmed the presence of recombinant protein. Conclusion: The fusion peptide produced in this research, is the first step in designing a universal influenza vaccine which needs to be assessed in animal models alongside proper control groups in future studies.