Optimization of recombinant Z (Junin virus)-N (measles)-GFP VLPs production

TRIFONE, C.A.; CALVENTE, N.I.; ARGÜELLES, M.H.; ESTEBAN, L.E.; CASTELLO, A.A.; LOZANO, M.E.; GLIKMANN, G.; TEMPRANA, C.F.

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

In the last decades, VLPs have been widely studied for the development of new vaccines. It has been previously described that VLPs can be formed when a fusion protein Z (from Junín virus)-GFP is expressed in 293T cells. Moreover a C-terminal fragment of the measles nucleoprotein (NCT) was cloned in frame with both Z and GFP in order to generate quimeric VLPs carrying the heterologous viral antigen. This work is mainly focused on the determination of optimal conditions for a laboratory scale VLPs production. pZ-NCT-GFP plasmid was transfected into Cos-7 cells. Transfected cell percentage was followed through flow cytometry at different times post transfection and remaining transfected cells after cell spliting and recombinant protein expression levels were also determined. In order to optimize VLPs production effectively using the transfection reagent, cell culture conditions and ultracentrifugation protocols, the VLP production scaling up was evaluated using roller bottles or T180 tissue culture flasks and different transfection/splitting strategies. Results obtained so far indicate that 168 h after cell spliting the transiently transfected cell population is still maintained up to 60 %. The best VLPs recovery was obtained from cell supernatant derived from a transfected T75 flask splitted into two T180 collected at day 3 after trasfection and day 4 after cell splitting.