Production of Nucleoprotein from Canine Distemper Virus in transplastomic tobacco plants

LAGUIA BECHER, MELINA; ANDREA TRENTINI; ROMANUTTI, CARINA; GALLO CALDERON, MARINA; MARIA ALEJANDRA ALVAREZ; JON VERAMENDI; PATRICIA MARCONI

Encuentro; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

PRODUCTION OF NUCLEOPROTEIN FROM CANINE DISTEMPER VIRUS IN TRANSPLASTOMIC TOBACCO PLANTSThe use of plants as bioreactors for the expression of pathogen antigens is an attractive alternative to produce cost-effective subunits vaccines. Chloroplast transplastomic technology offers many advantages over other plant expression systems, including stable high-level of recombinant protein expression and transgene containment. In this work, we evaluated the production of the nucleoprotein (NP) from an Argentina strain of canine distemper virus (CDV) in transplastomic tobacco plants. The NP sequence was fused to the endogenous promoter and 5′-untranslated region of the psbA gene and with a C-terminal 6xHis-tag. The transgene was targeted to the transcriptionally active spacer region between the trnI and trnA genes within the ribosomal operon. Finally, the construction was introduced into Nicotiana tabacum cv. Petite Havana chloroplasts by biolistic transformation. Stable integration of NP transgene and homoplasmy of the transformed plants were confirmed by Southern blot analysis. NP transplastomic plants exhibited normal growth and morphology. When total protein content from transplastomic leaf extracts was separated by SDS-PAGE, a 60-kDa band of the expected size for NP could be visualized upon staining with Coomassie blue. This band was also detected by Western blot using an anti-NP monoclonal antibody. In order to evaluate the solubility of NP, total soluble and insoluble proteins were extracted from developed mature leaves. The specific 60-kDa band was observed mainly in the insoluble fraction by Coomassie blue staining and Western blot. The chloroplast-derived NP will be used to evaluate the immune responses elicited in mice after subcutaneous and oral administration.