RODRIGUEZ TALOU Julian
congresos y reuniones científicas
Efficient production of a kdel-tagged Dengue virus protein in plant cell suspension cultures.
MARTÍNEZ, CA; GIULIETTI, AM; RODRIGUEZ TALOU, J
Puerto Madryn, Argentina
Congreso; XLVI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2010
Dengue virus envelope glycoprotein (DV-E) is the antigen associated with immunity induction and it is an effective candidate for the development of a subunit vaccine and a promising antigen for diagnostic kits. As a part of a project to develop a plant-made dengue virus vaccine, we explored the ability of plant cells to produce DV serotype 2 (DV-2) E protein in Nicotiana tabacum and Morinda citrifolia cell suspension cultures. DV-E cDNA was cloned with a signal peptide at its 5 end and with and without the addition of KDEL endoplasmic retention sequence at its 3 end to analyze its influence in recombinant protein accumulation levels. The expression cassette was sub-cloned into pCAMBIA 1305.2 binary vector and the cell suspension culture transformation was carried out using A. tumefaciens LBA4404. The maximum accumulation levels (0,71 ± 0,06 mg DV-E/L) were obtained by tobacco cells at 5 days of culture when KDEL tetrapeptide was fused. It represents 0,3% of the total soluble protein. Its integrity was confirmed by western blot. The recombinant protein was reactive with anti-E monoclonal and polyclonal antibodies. Our results demonstrate for the first time that plant cell in vitro cultures represents a low cost expression system suitable for the production of recombinant DV-E protein in which biosafety conditions are guaranteed.