Development of a dengue NS1 antigen expression system in mammalian cells.

MALNERO, C.M; MALIRAT V,; IBAÑEZ, I

Mar del Plata

Congreso; REUNION CONJUNTA SAIC, SAI, SAFIS, 2018; 2018

Sociedades de Biociencias: SAIC, SAI, SAFIS

The non-structural protein 1 (NS1) of dengue virus is a glycoprotein whose function has not been fully clarified. However, it is known to play a fundamental role in virus replication, immune evasion and pathogenesis. It can be detected in the bloodstream of patients with either primary or secondary infection at levels that correlate with viremia and disease severity. These characteristics point it as an interesting candidate for the development of diagnostic and therapeutic tools. Many attempts to express this protein in E. coli or in a yeast system result in insoluble aggregates with low protein yield and loss of biological activity, so they require refolding, involving optimization steps and time consumption.In this work an expression system based on mammalian cells was used to to obtain the NS1 Dengue virus protein in its native conformation and with conserved biological and antigenic characteristics. In addition, a simple purification step can be performed from the culture supernatant.The sequences corresponding to the NS1 of the four dengue serotypes were cloned into the pCAGGS vector downstream of the interleukin 2 secretion sequence and fused to a hexa histidine tag. The construct was transfected in HEK293-T cells and the expression was analyzed by Western Blot, after 48 and 72 hours, in supernatant and cell lysates. Subsequently, the assay was scaled up to larger production volumes and the protein was purified by affinity chromatography 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, recombinant NS1protein of the four dengue virus types (1-4) were successfully expressed and released to the culture medium in their native folded state, including post-translational modifications delivering optimal antigenicity (and making it suitable for use in vaccine research and serology-based assays). . These tools are promising for use in basic and applied research as well as antigens in diagnostic kits.