Expression of the ectodomain of the rabies virus glycoprotein G in insect cells for diagnostic purposes in vaccinated llamas

ALEXANDRA M. TARGOVNIK; GREGORIO MC CALLUM; MARIANA B. ARREGUI; LAUTARO F. BRACCO; MATÍAS MICUCCI; OSCAR PEREZ; MARÍA G. LÓPEZ; ALEJANDRO FERRARIO; MARÍA V. MIRANDA

Ginebra

Congreso; 18th European Congress on Biotechnology; 2018

Sociedad Europea de Biotecnología

Rabies is one of the principal zoonosis with worldwide distribution. The disease in llamas produces great economic losses in the productive system.The prevention against rabies virus (RABV) infection is done through vaccination with the inactivated virus. The level of protection can be assessedby determining the titre of neutralizing serum antibodies induced by RABV glycoprotein (RABV-G) in vaccinated individuals, which can bemeasured using an enzyme-linked immunosorbent assay. The aim of this work is to optimize a process for the production of the RABV-G ectodomainin Sf9 cell. The recombinant baculovirus was constructed by introducing the sequence corresponding to the ectodomain region of RABV-G, under thepolyhedrin promoter, fused to the viral signal peptide GP67 at its amino terminus and a 6-histidine tag at its carboxyl terminus. Despite the inefficientsecretion 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 Tunicamycintreatment confirmed that the protein is glycosilated. RABV-G was identified by LC-MS and was purified by metal ion affinity chromatographydirectly 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 serumsamples derived from rabies-vaccinated llamas. These results indicate that the recombinant RABV-G can be used as an antigen in the development ofdiagnostic kits.