Expression of a ricin B:F1:V fusion protein in tobacco hairy roots: steps toward a novel pneumonic plague vaccine

Recombinant subunit vaccines consisting of the F1 and V antigens of Yersinia pestis have shown remarkable promise for protection of animals and humans against bubonic and pneumonic plague. Previously we demonstrated that intranasal administration of a recombinant protein consisting of the ricin B chain (RTB) fused with an antigen purified from transgenic Nicotiana tabacum (tobacco) hairy root cultures was highly effective in stimulating a mucosal antibody response in mice (Medina-Bolivar et al., 2003). In the current study, transgenic tobacco plants and hairy roots were engineered to express a recombinant protein consisting of a hexahistidine-tagged RTB fused to tobacco sequence-optimized F1 and V genes (His:RTB:F1:V). Northern blot analysis of crude leaf extracts revealed that twenty-eight transgenic plant lines expressed a transcript of the predicted mRNA size detectable with the RTB probe. An RTB functional ELISA based on asialofetuin-binding activity was used to screen crude leaf protein extracts from these transgenic plants and select the two lines with the highest levels of fuctional RTB lectin activity. Analysis of the tissue localization of the recombinant mRNA in transformed plants and hairy roots developed from these plants suggested that His:RTB:F1:V expression levels were higher in hairy roots than in any tissue of the original transgenic plants. Asialofetuin-binding ELISA of transgenic hairy root extracts confirmed expression of functional RTB-containing fusion protein. Immunoblotting of lactose affinity-purified protein from hairy root lines using anti-RTB, anti-His-tag, and anti-V antibodies detected a protein of approximately 91 kDa, the predicted size of glycosylated His:RTB:F1:V. Purified His:RTB:F1:V protein will be used for intranasal immunization in mice, in which stimulation of mucosal immunity will be a first step towards an improved pneumonic plague vaccine.