Plant biotechnology approaches for a peptide vaccine design against Canine Distemper Virus.

GALLO CALDERÓN MARINA; ROMANUTTI CARINA; LAGUIA BECHER MELINA; ALVAREZ MARIA ALEJANDRA; MARCONI PATRICIA L

Congreso; IX Reunión Latinoamericana y del Caribe de biotecnología Agropecuaria y Forestal - IX Encuentro REDBIO.; 2016

Canine distemper virus (CDV) is an enveloped virus containing a non-segmented, single-stranded, negative RNA genome of approximately 15,000 nt. This genome encodes the following proteins: matrix, fusion, hemagglutinin (H), nucleocapsid (NP), polymerase and phosphoprotein. We have developed molecular methods to detect the glycoprotein H and NP viruses in rectal swab and clotted blood samples. Sequence analysis of the virus strains present in local samples showed that these strains are genetically distant from vaccine strains. In this work, we are reporting the establishment of Nicotiana tabacum cell cultures to produce H or NP proteins from CDV. We have already demonstrated the ability of N. tabacum whole plants to express heterologous proteins. The advantages of in vitro plant systems to produce possible biopharmaceuticals are, among others, their capacity to manage post-translational modifications, their culture medium simplicty, the absence of animal or human pathogenes and the feasibility of performing the productive process under GMP and GLP. Genes of full length H and NP of local and vaccine strains were amplified by RT PCR. The primers used in the amplification reaction include the Kozak sequence and S2S sequence at ´5 end. The amplified product were digested, purified and cloned into pENTR4 vector. Finally, these constructs and the pK7WG2 binary vector were recombined using the GATEWAY technology (Invitrogen). We obtained several constructs carrying the H or NP coding regions that were introduced into Agrobacterium tumefaciens EHA101 competent cells by electroporation. The transgenes were transformed into the tobacco plants (Nicotiana tabacum) to express the heterologous proteins.