Bacillus subtilis as a Host for Recombinant Hemagglutinin Production of the Influenza A (H5N1) Virus

Abstract Background and Aims: Influenza A(H5N1) viruses  circulating in animals might evolve and acquire the ability to spread from  human to human and thus start a pandemic. Hemagglutinin (HA) has been shown to play a major role in binding of influenza virus to its target cell and the main neutralizing antibody responses elicit against this region. Recent studies have shown that glycosylation of HA is not necessary for its immunogenicity. Bacillus subtilis has been identified as a free endotoxin host for high expression and secretion of heterologous proteins with immunological activity. This bacterium is not capable of supporting glycosylation process. However, it could be an appropriate host to produce new recombinant HA1 for vaccine research purposes. In this study we constructed a recombinant B. subtilis that was able to express and secrete HA1 protein into cytoplasmic and extracellular medium. Materials and Methods: HA1 gene was amplified and cloned into pGEM® 5Zf(–) vector. It was then subcloned into shuttle vector PHT43 and transferred to E.coli for propagation. Accuracy of PHT43-HA1 construct was confirmed by sequencing and restriction map. The recombinant plasmid was extracted from E.coli and used to transform of B.subtilis by electroporation. Following IPTG induction, the total cell protein and the protein secreted into media were analysed through a time course using SDS-PAGE. Results: The accuracy of  PHT43-HA1 construct was confirmed by sequencing and enzymatic digestion analysis. SDS-PAGE results showed that the recombinant HA1 protein was successfully expressed and secreted into medium. Conclusion: The HA1 protein produced here could be considered and evaluated as a protective antigen which its immunogenicity potential needs to be assessed in animal models along with proper control groups.