 Producción de antigenos y anticuerpos de interés veterinario en plantas transplastómicas de tabaco

EZEQUIEL MATIAS LENTZ; MARINA MOZGOVOJ; DEMIAN BELLIDO; MARIA EUGENIA SEGRETIN; GARAICOECHEA LORENA; PARREÑO V; DUS SANTOS MARIA JOSE; ANDRES WIGDOROVITZ

Congreso; Biotecnología Habana 2011; 2011

In this work, we evaluated the feasibility of using transplastomic plants (Nicotiana tabacum) for the production of proteins of interest in veterinary medicine: two viral antigens and a camelid single-chain antibody (VHH). The first protein produced in this system was a translational fusion of an immunogenic peptide from VP1 of foot-and-mouth disease virus and the enzime -glucuronidase (VP-GUS). Transplastomic plants engineered to express the aforementioned construction were characterized at the molecular level. The heterologous protein accumulated up to 51% of the total soluble leaf protein (TSP), or 8 mg/g of fresh tissue (FT), while retaining its enzymatic activity and immunogenicity. The observed expression levels were clearly higher than those obtained previously in nuclear transgenic plants. Despite these high levels of expression, the phenotype of the transplastomic plants was indistinguishable from that of wild-type plants. This proof of concept with encouraging results, allowed us to continue evaluating this system for the production of other recombinant proteins. The second antigen expressed was VP8*, a non-glycosilated capsid protein from bovine rotavirus (BRV). VP8* formed insoluble aggregates in the stroma and was partially solubilized by sonication. The total plant extract contained 600 g of VP8* g/g FT, and only 150 g/g FT of VP8* could be solubilized after 3 sonication pulses. The protein from both fractions was immunogenic and conferred protection against the virus in the suckling mouse model. The third molecule expressed was the VHH clone 3B2 directed against a conformational epitope of the VP6, an inner capsid protein of BVR. This nanobody was expressed using three strategies: accumulation in the stroma by itself (VHH), as a fusion to the -glucuronidase (GUS-E-VHH), and targeting the VHH to the thylakoid lumen (pep-VHH). In VHH transplastomic plants the protein accumulated at very low levels, almost undetectable by western blot. Expression levels were improved in both pep-VHH (2,5% of TSP; 60 g/g FT) and GUS-E-VHH plants (3 % of TSP; 70 g/g TF), but in the latter case degradation products were observed. Homoplastic pep-VHH plants had a chlorotic phenotype, which was more intense when plants were grown under higher illumination. These results show the difficulty of expressing this VHH in chloroplasts and suggest a toxic effect of the transgene in the plant. The targeting to the lumen of thylakoids favors the stability of the heterologous protein, which is consistent with its successful expression in E. coli when it is directed to the bacterial periplasm. The results presented in this work support the use of transplastomic plants as biorreactors, in order to achieve feasible industrial alternatives