Secretory expression of hemagglutinin globular domain (HA1) of the influenza A (H5N1) virus in Bacillus subtilis

Abstract

Background and Aim: H5N1 influenza viruses may acquire the ability to transmit between humans thus, there is the possibility of a pandemic event. HA1 protein has been shown to play a major role in binding the virus to its target cell and the main neutralizing antibody responses elicit against this region. Bacillus subtilis has been identified as a free endotoxin host for expression and secretion of heterologous proteins with immunological activity. Although the bacteria is not capable of making glycosylated proteins, it has been shown that glycosylation of HA is not much necessary for its immunogenicity. Here we produced secretory recombinant HA1 protein in B. subtilis. Materials and Methods: HA1 sequence was cloned into pGEM® 5Zf(–)vector. It was then subcloned into shuttle vector PHT43 and transferred to E.coli for replication. The recombinant plasmid was extracted from E.coli and used to transform of B.subtilis by electroporation. After induction of recombinant B.subtilis by IPTG, 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. Conclusions: B.subtilis is a free endotoxin host which could be a favorite prokaryotic platform for production of the recombinant HA1 protein.The HA1 protein produced here could be considered and evaluated as a candidate vaccine which its immunogenicity potential needs to be assessed in animal models along with proper control groups.

Keywords

• Bacillus subtilis
• secretory expression
• influenza
• H5N1