Figure 1: Generation and characterization of tM2e VLPs. (a) Diagram of tM2e construct. (b) Tetramerization of tM2e in VLPs. M2e VLPs were produced as described in Material, and Methods. One μg of tM2e VLPs was cross-linked with BS3 at concentrations of 4 mM. Cross-linked tM2e VLPs samples were applied to Western blot and probed using mouse monoclonal antibody 14C2 (Abcam Inc., Cambridge, MA). Lane 1: without BS3. Lane 2: with 4 mM BS3. (c) Cell surface expression of membrane-anchored tM2e. Surface expression of the membrane-anchored tM2e was detected by cell surface biotinylation. Lane 1: Cell lysate from cells infected with rBV expressing membrane-anchored tM2e; Lane 2: mock rBV (rBV expressing human immunodeficiency virus Gag)-infected cells. ((d), (e)) Optimization of VLP production: Sf9 cells were infected with rBVs expressing tM2e, M1, and tFliC at different MOIs as designated at the bottom. VLPs were prepared as described in Materials and Methods. The resulting VLPs were analyzed by Western blotting. tM2e and M1 bands were probed with mouse monoclonal antibody 14C2 and mouse monoclonal anti-M1 antibody. Membrane-anchored truncated flagellin (tFliC) was probed with guinea pig anti-flagellin polyclonal antibody. The asterisk means MOIs of different virus. The best ratio was framed in the figure. (f) Western blotting of tM2e VLP and recombinant M2e protein. Recombinant M2e protein (Lanes 1, 2, and 3 and 4, 15, 30, 60, and 120 ng, resp.), tM2e VLPs (Lane 6: 5 μg total protein) and tM2e/tFliC VLPs (Lane 5: 5 μg totoal protein) were loaded and detected by Western blot using mouse anti-M2e monoclonal antibody (14C2). Amount of M2e protein incorporated in tM2 VLPs was calculated by spot densitometry analysis using serial diluted recombinant tM2e protein as a standard.