Characterization of purified mammalian VLPs.

<p>The total proteins of purified H3N2-VLPs and H3N2 virus (A) and H5N1-VLPs and H5N1 virus (C) were resolved by SDS-PAGE in a 7.5–17.5% gradient gel and stained with Coomassie blue. Molecular masses of protein markers are shown on the right. Each relevant band, as marked with an arrow and a number, was subjected to LC/MS/MS analysis to identify its composition. The identified viral protein bands of H3N2- and H5N1-VLPs (marked with parentheses in panels A and C) confirmed by Western blot analyses with relevant specific antibodies for H3N2-VLPs and virus (B) and for H5N1-VLPs and virus (D). The relative abundances of HA and NA, quantified by Chemigenius 2 (SYNGENE, Frederick, MD) and GeneTools (version 3.07) software, were 4∶1 for H3N2-VLPs and 3∶2 for H5N1-VLPs. The HA protein attributes to 12.8% and 18% of total proteins in H3N2- and H5N1-VLPs, respectively. (E) Assessment of HA function by hemagglutination assay. The amounts of mammalian VLPs or virus used are indicated, in a twofold serial dilution. TNE (buffer of VLPs) and PBS were included as the negative controls in the hemagglutination assays.