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Zusammenfassung:
Influenza is a global, highly contagious disease causing several million human infections every year. The most effective means of controlling seasonal influenza outbreaks is prophylactic vaccination. Current inactivated influenza vaccines contain either whole influenza viruses or viral membrane components. So called subunit vaccines for example consists mainly of the transmembrane glycoprotein hemagglutinin (HA), which is regarded as the major immunogen of the influenza virus. In the present study a method for the purification of HA from viral and host cell (Madin Darby canine kidney (MDCK)) membranes is presented. Extensive studies were done using different human influenza virus strains including two strains from the season 2007/08 (A/Wisconsin/67/2005 (H3N2), B/Malaysia/2506/2004) and A/Puerto Rico/8/34 (H1N1). After chemical inactivation (beta-propiolactone) of the culture broth the viral and host cell membranes were solubilized by a combination of different detergents. Remaining particles were removed by sequential centrifugation and filtration. Subsequently, the supernatant was diafiltrated against phosphate buffered saline to remove salts and free detergents. Further depletion of detergents in the broth was achieved by hydrophobic interaction chromatography using Amberlite XAD-4 as a resin.
Purification was performed by a combination of two affinity chromatography steps using (1) sulfated reinforced cellulose membrane adsorbers and (2) column based lectin-affinity chromatography. As a final polishing step size exclusion chromatography was included to the process. The recovery of HA based on single radial immunodiffusion (SRID) as well as the purity by the means of non-reducing SDS-PAGE were analyzed. From the results it can be concluded that the suggested method is suited for the purification of HA of cell culture derived influenza virus without isolating the viral particles from cultivation broth.
