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

GALLO CALDERÓN, MARINA; ROMANUTTI, CARINA; LAGUÍA BECHER, MELINA; ALVAREZ, MARÍA ALEJANDRA AND MARCONI, PATRICIA L.

Lima

Congreso; Novena Reunión Latinoamericana y del Caribe de biotecnología Agropecuaria y Forestal, IX Encuentro REDBIO 2016; 2016

Canine distemper virus (CDV) is an enveloped virus containing anon-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 NPviruses in rectal swab and clotted blood samples. Sequence analysis ofthe virus strains present in local samples showed that these strains are geneticallydistant from vaccine strains. Inthis 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 ofN. tabacum whole plants to expressheterologous proteins. The advantages of invitro plant systems to produce possible biopharmaceuticals are, amongothers, their capacity to manage post-translational modifications, theirculture medium simplicty, the absence of animal or human pathogenes and thefeasibility of performing the productive process under GMP and GLP. Genes of fulllength H and NP of local and vaccine strains were amplified by RT PCR. Theprimers used in the amplification reaction include the Kozak sequence and S2Ssequence at ´5 end. The amplified product were digested, purified and cloned intopENTR4 vector. Finally, these constructs and the pK7WG2 binary vector were recombinedusingthe GATEWAYÒ technology (Invitrogen). We obtained severalconstructs carrying the H or NP coding regions that were introduced into Agrobacterium tumefaciens EHA101 competent cells byelectroporation. The transgenes were transformed into the tobacco plants (Nicotiana tabacum) to express theheterologous proteins.