CONICET | Buscador de Institutos y Recursos Humanos

Dengue is animportant viral disease transmitted by mosquitoes, and in recent years it hasbecome endemic in Argentina. High levels of the Dengue virus protein NS1 hasbeen detected in the sera of infected patients, and for this reason thisprotein has been used for the development of diagnostic kits. Nanoantibodies (NAbs),which are fragments of heavy chain antibodies obtained from camelids, are apotentially useful tool for Dengue detection. These small molecules typically exhibitstrong specificity and affinity for different antigens, are thermostable and canbe easily produced in microorganisms at a low cost. Most importantly they canbe straightforwardly modified by standard molecular biology techniques. Giventhe need for innovative and affordable dengue detection systems, here we reportthe production of NAbs with ability to recognize the NS1 protein of Dengue virus.For this purpose, twollamas were immunized with purified and inactivated supernatant of Dengue infectedcells containing high levels of NS1 protein. Total RNA was isolated fromperipheral blood. After cDNA synthesis, part of the genes encoding antibodyheavy chains were amplified by PCR and cloned into the pHEN4 phage displayvector. This plasmid was transformed into TG1 E. coli cells, which were later infectedwith the M13K07 helper phage. Selection of NS1 specific binders was carried outby phage display. NAbs were separated from TG1 periplasm by osmotic shock, andspecific binding to purified NS1 was tested by ELISA.Two libraries of about2x108 transformants with more than 88% of inserts of the right size wereobtained. After 3 rounds of panning, 192 candidate clones were isolated andamplified in TG1 cells. Periplasmic extraction of NAbs allowed us to determinethat isolated candidates specifically recognize NS1 protein of Dengue virus. Selectedclones will be modified and produced at a large scale to generate a Dengue NS1 detectionkit.

enviar mensaje