Expression of recombinant VP2 in N. benthamiana.

A) Schematic representation of pBI-His-pVP2 and p35:AMCV-P19 constructs used for plant agroinfiltration. 35S: CaMV 35S promoter; Ω: TMV translational enhancer sequence; His-pVP2: sequence encoding the first 466 aa of the pVP2 protein N-terminally fused to His-tag; P19: sequence encoding the AMCV p19 gene-silencing suppressor; nos-t: nopaline synthase gene terminator. B) Evaluation by ELISA of His-pVP2 expression in agroinfiltrated leaf extracts at different time points (5, 6, 7, 8 DPI). Fifty μg of TSP extracted from agroinfiltrated leaves were distributed in triplicate into the wells and His-pVP2 was detected with an anti-VP2 antibody. The reported values are the mean of three independent experiments, and error bars represent SD of the means. C+: inactivated IBDV; C-: p19 plant extract. C) Western blot analysis of His-pVP2 expression in leaves extract at 7 DPI. Ten μg of TSP were separated by 11% SDS-PAGE and His-pVP2 was detected using an anti-VP2 serum. C+: inactivated IBDV; C-: P19 agroinfiltrated leaf extract. His-pVP2: His-pVP2 agronfiltrated leaf extract. The arrow indicates the band corresponding to His-pVP2 while the asterisk a possible degradation product.