INVESTIGADORES
IBAÑEZ Lorena Itati
congresos y reuniones científicas
Título:
DEVELOPMENT OF A DENGUE NS1 ANTIGEN EXPRESSION SYSTEM IN MAMMALIAN CELLS.
Autor/es:
MALNERO C; MALIRAT V; IBAÑEZ LI
Lugar:
Mar del Plata
Reunión:
Congreso; Reunión Conjunta SAIC SAFE SAI; 2018
Institución organizadora:
SAIC
Resumen:
The nonstructural protein 1 (NS1) of dengue virus is a multifaceted glycoprotein known to play a fundamental role in virus replication, immune evasion and pathogenesis. It can be detected in the bloodstream of patients, with both primary or secondary infections, and its levels correlate with viremia and disease severity. These characteristics point it as an interesting target for the development of diagnostic and therapeutic tools. Most attempts to express this protein in Escherichia coli or in a yeast system have resulted in insoluble aggregates with low protein yield and loss of biological activity. In addition, refolding of the aggregates requires time consuming optimization steps.In this work an expression system based on mammalian cells was developed to produce the protein in its native conformation and with intact biological and antigenic characteristics through a simple purification step from the cell culture supernatant. DNA fragments encoding NS1 protein from the four dengue serotypes were cloned in pCAGGS vector by the infusion PCR cloning system, downstream of the interleukin 2 secretion sequence and fused to a hexa histidine tag. Protein expression in HEK293-T cells culture supernatant and lysate was analyzed by Western Blot 48 and 72 hours after transfection. Subsequently, the assay was scaled up to larger production volumes and the protein was purified by affinitychromatography using a Ni-NTA resin (Amintra?). In order to assess antigenicity of serotype 1 purified protein, an ELISA with serum samples from infected and healthy individuals was carried out. In conclusion, it was demonstrated that the recombinant NS1 proteins of the four serotypes are correctly expressed and released to the culture medium with proper antigenicity. These tools are promising for use in basic research as wells as for the production of antigens for diagnostic kits.