High level expression and purification of hemagglutinin subtype H9 of influenza virus

Influenza virus is a respiratory pathogen having high mutation rate, thus displaying a great antigenic diversity. The frequent mutations of the influenza viral surface proteins, hemagglutinin and neuraminidase, allow the virus to evade the host immune system, resulting in annual epidemics and occasional pandemics. Vaccination is the most effective way to protect against influenza virus infections. Currently, licensed vaccines are grown in chicken eggs and the process of making a new vaccine based on newly circulating viruses is quite laborious. Future vaccine strategies are therefore focused on increasing capacity, reducing cost and time required for antigen production. Subunit vaccines like recombinant hemagglutinin offer an alternative over conventional vaccines. The current study shows designing of an antigen to get high level of secretory expression and stabilized native trimer structure. H9HA was assembled with sequence for the tissue plasminogen activator signal peptide (tPA-SP) and foldon domain of bacteriophage T4 fibritin (FT) using splicing by overlap extension amplification. The amplified product was cloned in pCMVR-8kb having human cytomegalovirus (CMV) immediate-early enhancer and promoter for transient expression of HA in 293F cells. SDS PAGE analysis confirmed a band of ≃70 kDa corresponding to H9HA which was further confirmed by western blot analysis. Supernatants having recombinant H9HA were pooled together and passed through Ni-NTA agarose column for purification purposes. The purified recombinant protein was found biologically active. We describe the design of an antigen and cloning technique that allow transient expression of gene under control of the CMV promoter. The findings from our studies will be helpful in the optimal design of the HA based antigen for Influenza vaccines. Furthermore, immunization study and systematic analysis of the cross protection profiles among HA antigens is needed to select an antigen providing broad range p- otection.