Production of bacterial antigens in plant expression system

The aim of this study was to evaluate tobacco plants, transformed by stable agroinfection, as expression system of bacterial antigenic proteins as a model of edible vaccine for swine. The attention was also focalized on the analysis of tobacco progeny in order to assess the stability of the bacterial genes into the plant genome. Three independent lines of transgenic tobacco plants (Rossi et al., 2003a; Rossi et al., 2003b), respectively expressing the F18 adhesive fimbriae and VT2e B-subunit genes from Verocytotoxic Escherichia coli strains and the flgK flagellin from Salmonella typhimurium strain were previously obtained by Agrobacterium-mediated stable transformation technique (Rossi et al., 2013; Rossi et al., 2014a). The chimeric constructs pBIpGLOB-F18, pBIpGLOB-VT2eB, pBIpGLOB-flgK were used to transform Agrobacterium tumefaciens strain EHA105. The GLOB promoter is the soybean basic 7S globulin promoter (DDBJ no. AX006477) and was used for the seed-specific protein expression (Reggi et al., 2005; Rossi et al., 2014b). The entire genes, codifying respectively for F18, VT2e-B and flgK, were amplified from genomic DNA of transgenic plants by PCR (table 1). The mRNA was evaluated by Northern blot analysis on the seeds 12 days after pollination. The total proteins were extracted from all mature transformed tobacco lines by homogenization with liquid N2 in a mortar with the solubilization buffer (50 mM Tris, pH 8, 5 mM EDTA, 200 mM NaCl, 0.1% Tween 20) and were estimated by a Bradford assay (using bovine serum albumin as the standard). The expression of each antigen in the total protein samples was evaluated by Western blotting with specific polyclonal antibodies. The R1 and R2 generations, propagated in a greenhouse were evaluated using the same experimental conditions previously described. The presence of genes codifying for the antigenic proteins in the genome of the three transgenic plants were confirmed by PCR. Transgenes were identified by electrophoresis on agarose gel by the presence of an amplified product of 0.5 Kb, representing the gene encoding F18 fimbriae , by the presence of an amplified product of 0.25 Kb, representing the gene encoding VT2e-B and by the presence of 1.6Kb, representing flgK. Northern blot and Western blot analyses detected specific signals in all samples, and by comparison with positive controls, the amount of antigenic proteins was estimated to be about 0.6 mg per gram of seeds corresponding to 0.33% of the total soluble protein in tobacco seeds. For each line the obtained amount of antigens is sufficient for subsequent oral immunization trials (Verdonck et al. 2007, Lamphear et al. 2002,). Obtained data showed the inheritance of transgenes in the R0, R1, R2 generations and the stable integration of VT2e-B, F18 and flgK genes into tobacco genome.