Modification and improvement of a plasmid vector for the production of antigenic molecules in GM tobacco, for veterinary use.

The production of important molecules (as subunit vaccines) in plants is increasingly considered for relevant advantages: low costs of production, purification and delivery, no risks of contamination by pathogens and high scale production, but improvement and enhancement of transformation techniques are needed. MAR/SARs (Matrix/Scaffold Attachment Regions) have been reported as a network of proteinaceous fibrils that permeates the nucleus and organizes chromatin into a series of topologically isolated loop domains of 5-200 kb. These sequences may influence the structure of transgenes and their expression possibly reducing or eliminating some forms of gene silencing. Our research is addressed at the production of plant derived antigens to be used in veterinary prophylaxis. In this field, the optimisation of transgene expression is crucial, also because of the necessity of plant containment during the whole cultivation period. In particular, we sub-cloned Rb7, a MAR sequence from Nicotiana tabacum,, in the binary vector pAMPAT, inside LB and RB t-DNA terminations, in its two possible orientations. The vector expression cassette carries a 450bp portion of Fib, Fibrinogen Binding Protein, from Staphylococcus aureus, under the control of 35SS constitutive promoter. The Fib protein fragment was proved to be effective against S. aureus mastitis in dairy cattle. Nicotiana tabacum, var. Samsun was transformed via Agrobacterium tumefaciens with the four constructs carrying Rb7 elements in all their possible combinations. Statistical analysis was performed after four different experiments, showing an enhanced transformation efficiency for MAR containing constructs (higher shoot number and shorter shooting time). PCR analysis on genomic DNA and on retro-transcribed RNA, from transformed leaves, confirmed the transgene presence and transcription. Protein extraction was performed 40 Molecular and immunological analysis on transformed plants are now in progress, to define the transgene copy number and the resulting protein expression level.