Fig. 5
Enhanced expression of vaccine antigen proteins using newly developed PVX-based vectors in N. benthamiana. (A) Schematic diagram of PVX-derived vaccine vectors in which the GFP-coding sequence of pP3P38:GFP and pP3NSs: GFP was replaced with the gene encoding VP1 (BiP: VP1:CBD: HDEL) or S2 (BiP: S2:His6:HDEL). (B) Western blot analysis of soluble proteins (20 µg per sample) extracted from agroinfiltrated N. benthamiana leaves expressing VP1 or S2, which were detected using an anti-CBD or anti-His6 antibody, respectively. Band intensities were quantified using ImageJ. Data represent the mean ± SD from at least three independent experiments (n = 3). Statistical significance was determined using the Student’s t-test. Asterisks indicate significant differences compared with pPVX: VP1 or pPVX: S2 (*p < 0.05, **p < 0.01, and ***p < 0.001). (C) To confirm antigen identity, the same membranes were stripped and reprobed with anti-CBD or anti-His₆ antibodies after initial detection with commercial anti-FMDV (VP1) and anti-S2 (S2) antibodies, respectively. Samples were collected at 3 and 5 dpi, and total soluble proteins (20 µg per sample) were separated by SDS-PAGE prior to immunodetection. The Rubisco large subunit stained with PS served as a loading control. GFP-expressing samples were included as negative controls (NC).
