Baculoviral Co-Expression of HA, NA and M1 Proteins of Highly Pathogenic H5N1 Influenza Virus in Insect Cells

Abstract

Background:: Highly pathogenic H5N1 influenza virus is causing damages to the poultry industry and is responsible for loss of human lives. Vaccination is the most effective method to prevent and control influenza infections. Recombinant virus-like particles (VLPs) by baculovirus expression vector system have been suggested as a promising platform for new viral vaccines. Objectives:: In this study we constructed a recombinant baculovirus that was potent to express influenza structural proteins; Haemagglutinin (HA) and Neuraminidase (NA) as well as matrix protein (M1) which is essential to generate immunogenic VLPs in insect cells. Materials and Methods:: A triplet cassette providing simultaneous and independent expression of HA, NA and M1 genes of avian influenza virus (A/Indonesia/5/05(H5N1)) was designed and subjected to synthesis. The cassette was then cloned into pFastBac1 plasmid and then transformed in to competent Escherichia coli DH10Bac cells and the recombinant bacmids were produced following the homologous Tn7 site-specific transposition. This construction was verified by polymerase chain reaction (PCR) and then transfected into Sf9 insect cells to package new recombinant baculovirus expressing complex proteins of the interest. Results:: Restriction map of pFastBacIHNM1 plasmid confirmed the fidelity of the clone. The PCR carried out on the recombinant bacmids as template indicated that a proper homologous recombination has occurred between pFastBacIHNM1 donor plasmid and the bacmid. Following the transfection of new recombinant bacmids to Sf9 cells, cytopathic effects were observed which indicating the successful packaging of recombinant baculovirus. Protein analysis of the infected Sf9 cells showed that all target proteins were efficiently expressed at the same time. Conclusions:: The recombinant baculovirus constructed in this work has proper characteristics to produce H5N1 influenza virus-like particles in Sf9 cells.