FIG. 4.

Assembly of SCoV 7a protein into VLPs. Initially, 3 μg of pCAGGS-S, 3 μg of pCAGGS-M, 3 μg of pCAGGS-N, 0.5 μg of pCAGGS-E, and 2 μg of pCAGGS-7a with 1.5 μg of pCAGGS-3a (3a+) or 1.5 μg of pCAGGS empty plasmid (3a−) were transfected into subconfluent 293T cells. At 48 h posttransfection, media were harvested and clarified and the released VLPs were pelleted by centrifugation through a 20% sucrose cushion. Cell extracts were also prepared at 48 h posttransfection. SCoV VLPs were resuspended in NTE buffer containing 0.3% of BSA and immunoprecipitated with mouse anti-SCoV S monoclonal antibody. The captured SCoV VLPs (VLP) and cell lysates (Cell lysate) were analyzed using Western blot analysis with anti-7a antibody (7a), anti-S protein antibody (S), anti-M protein antibody (M), anti-N protein antibody (N), and anti-3a antibody (3a).