MIRANDA Maria Victoria
congresos y reuniones científicas
Expression of the ectodomain of the rabies virus glycoprotein G in insect cells for diagnostic purposes in vaccinated llamas
A. TARGOVNIK; MC CALLUM GREGORIO; MARIANA ARREGUI; LAUTARO BRACCO; MICUCCI; O PEREZ; GABRIELA LOPEZ; VICTORIA ALFONSO; FERRARI ALEJANDRO; MARIA VICTORIA MIRANDA
Congreso; European Federation of Biotechnology; 2018
Rabies is one of the principal zoonosis with worldwide distrIbution. The disease in llamas produces great economic losses inthe productive system. The prevention against rabies virus (RABV) infection is done through vaccination with the inactivated virus. The level of protection can be assessed by determining the titre of neutralizing serum antibodies induced by RABV glycoprotein (RABV-G) in vaccinated individuals, which can be measured using an enzyme-linked immunosorbent assay. The aimof thiswork is to optimize a process for the production of the RABV-G ectodomain in Sf9 cell. The recombinant baculovirus was constructed by introducing the sequence corresponding to the ectodomain region of RABV-G, under the polyhedrin promoter, fused to the viral signal peptide GP67 at its amino terminus and a 6-histidine tag at its carboxyl terminus. Despite the inefficient secretion of the RABV-G, it was possible to recover it from the cell lysate. Insect cells infected at multiplicity of infection of 0.5 at day 3 expressed 7mg litre-1. The recombinant RABV-G had a molecular mass of approximately 50 kDa according to SDS-PAGE analysis. PGnase F and Tunicamycin treatment confirmed that the protein is glycosilated. RABV-G was identified by LC-MS and was purified by metal ion affinity chromatography directly from the cell extract with a yield of 99% and a purity of 67%. Purified RABV-G was succesfully used to detect specific antibodies in serum samples derived from rabies-vaccinatedllamas. These results indicate that the recombinant RABV-G can be used as an antigen in the development of diagnostic kits.