Secretory expression of all 16 subtypes of the hemagglutinin 1 protein of influenza A virus in insect cells

► Expression of all 16 subtypes of HA1 of influenza A viruses. ► Development of an efficient and simple purification process. ► Potential for large scale production. ► Potential application in immunoassays. Influenza viruses are known for their ability to change their antigenic structure and create new viral strains. Hemagglutinin (HA), for which 16 subtypes have been identified, is a major antigenic determinant essential for the pathogenesis of influenza A viruses. To predict and monitor future epidemics, it is critical to produce various HA subtype antigens conveniently and rapidly. A simple, effective, and economic method to generate subunit HA1 of all 16 HA subtypes as recombinant proteins (rHA1) is reported. rHA1 proteins are expressed in insect cells as secretory proteins after integration into a baculovirus expression vector containing a 6 × His tag element and the signal peptide of the GP67 protein, a membrane glycoprotein identified in Autographa californica nuclear polyhedrosis virus. The proteins can be purified to ≥90% purity using a single Ni 2+-chelating affinity chromatography step, yielding a recovery rate of about 50%. The rHA1 proteins elicit high titer antibodies in mice and show high specificity in Western blots. This study paves the way for subtype specific detection methods and for future studies of the immune relationships among the subtypes of influenza A virus HA proteins.