A Novel System for Rapid and Cost-Effective Production of Detection and Diagnostic Reagents of West Nile Virus in Plants
Western blot analysis of WNV DIII antigen and E16 mAb expression in N. benthamiana and lettuce. (a) WNV DIII. Leaf protein extracts were separated on a 4–20% SDS-PAGE gradient gel and transferred onto PVDF membranes. The membranes were probed with a rabbit anti-WNV DIII polyclonal antibody. Lane 1: E. coli-derived DIII standard; lane 2: protein extract from leaves infiltrated with buffer (negative control); lane 3: extract from DIII construct infiltrated N. benthamiana leaves. (b) and (c) Expression of E16. Wild-type laboratory-grown lettuce or N. benthamiana were infiltrated with dual-replicon geminiviral vector pBY-HL (hE16-no-KDEL). R and harvested on 4 dpi. Total leaf protein extracts were separated on 4–20% SDS-PAGE gradient gels under reducing conditions and transferred to PVDF membranes. The membranes were incubated with a goat anti-human-gamma chain antibody to detect HC (b) or a goat anti-human-kappa chain antibody to detect LC (c). Lane 1: human IgG reference standard; lane 2: extract from lettuce leaves infiltrated with buffer (lettuce negative control); lane 3: protein samples from lettuce infiltrated with geminiviral vector pBY-HL(hE16-no-KDEL). R; lane 4: extract from buffer-infiltrated N. benthamiana leaves (negative control); Lane 5: N. benthamiana leaf protein extract infiltrated with pBY-HL(hE16-no-KDEL). R vector.
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