EXPRESIÓN DE LA GLICOPROTEÍNA DEL VIRUS RÁBICO EN Lactococcus lactis.

Abstract

Rabies is an acute, progressive, incurable and deadly infectious disease with worldwide distribution. It enters and cripples the host nervous system in humans and other mammals. It is caused by a single negative-strand RNA virus of 11-15 kb size, from the Rhabdoviridae family, Lyssavirus genus. The route of infection is usually by a bite from an infected animal. The principal rabies virulence protein involved in the humoral immune response is Glycoprotein G (Gp-G). Antibodies against Gp-G block experimental infections in vitro, and Gp-G protein is incorporated in viral vectors to create recombinant rabies vaccines. Gram-positive Lactococcus lactis is non-pathogenic and generally considered safe (GRAS) for human consumption. Its protein expression, transport and wall-anchoring genetic mechanisms have been characterized. One of them, mediated by the enzyme sortase, has been exploited for the cell-wall anchoring of recombinant proteins expressed in L. lactis, and recombinant antigens have been expressed both in the cytoplasm and the cell wall of the bacteria. Several research groups are working in research and development for heterologous protein expression in L. lactis. A 1500 bp Gp-G DNA sequence was amplified from plasmid pKB3- JE-13-Gp-G with primers GPA1 For and GPA 1 Rev by PCR (polymerase chain reaction). Both GPA1 For and GPA 1 Rev had SacI and XmaI restriction sites added in their 5' ends. The 1500 bp amplified Gp-G fragment was then inserted in plasmid vector pGEMT and cloned into E. coli Top 10. Gp-G was then inserted into L. lactis expression plasmid pOri23. Both pGEMT-Gp-G plasmid and pOri23-CL- plasmid were digested with XmaI and SacI, and the 1500 bp Gp-G fragment from pGEMT was observed in an agarose gel, extracted and ligated into pOri23-CL-, which then was used to transform E. coli Top 10 competent cells. Cloning success was verified by PCR and restriction digestion, producing a 6.6 kb band from digested pOri23-CL- and 1.5 kb band from Gp-G. Sequencing of the resulting plasmid pOri23-Gp-G also confirmed cloning success. In this work wild type and two recombinant L. lactis C3-1 and C2-1 expressing Gp-G were used. Indirect immunofluorescence experiments were performed with confocal microscopy to evaluate Gp-G expression using commercial monoclonal anti-Gp-G antibodies and polyclonal anti-Gp-G antibodies. Both C3-1 and C2-1 were recognized with both monoclonal and polyclonal antibodies, although signal strength was lower in C2-1 compared with C3-1. ELISA experiments performed with the monoclonal antibody in L. lactis C3-1, C2-1 and wild type in 10-30 μg/mL antigen concentration resulted in a strongest signal for C3-1 at 30 μg/mL. In conclusion, we showed that Rabies Gp-G protein is expressed in L. lactis, with higher levels of expression in C3-1 compared with C2-1 and with L. lactis wild type observed by indirect immunofluorescence and ELISA assays. This results is also confirmed after quantification of Gp-G expression in C3-1 supernatant. The product obtained from this work can be used as a candidate vaccine in an animal model of rabies infection, to evaluate the level of protection that can be provided by immunization in an infectious challenge.