FIG 4.

Characterization of protease-dependent and protease-independent PV-1 SC6b VLPs. (A) Immunoblot for PV VP1, VP0, 6xHIS and 2A peptide. Peak gradient fractions for each VLP preparation were concentrated using 100 kDa microcentrifuge concentrators (Amicon) to ~100 μL. All samples were mixed 1:1 with 2x Laemmli buffer, boiled and separated by SDS-PAGE prior to analysis by immunoblot using mouse monoclonal α-VP1, a rabbit polyclonal α-VP0, mouse monoclonal α-HIS and mouse monoclonal α-2A. (B) Antigenicity of concentrated PV-1 VLPs. Reactivity of concentrated fractions with Mab 234 (D-antigen) and Mab 1588 (C-antigen) in ELISA. OD at λ = 492 nm is represented in arbitrary units (n = 3). Means +/− standard deviation. Statistical analysis determined by two-tailed T-test (ns, not significant; *, P value > 0.05, ***, P value > 0.001). Construct nomenclature is as follows: 31-0 = VP3 P2A VP1 VP0, 30-1 = VP3 P2A VP0 VP1, 10-3 = VP1 P2A VP0 VP3 & 13-0 = VP1 P2A VP3 VP0.