RESUMEN
Bovine herpesvirus (BoHV) glycoprotein E (gE) is a non-essential envelope glycoprotein and the deletion of gE has been used to develop BoHV-1 and BoHV-5 differential vaccine strains. The DIVA (Differentiation of Infected from Vaccinated Animals) strategy, using marker vaccines based on gE-negative BoHV strains, allows the identification of vaccinated or infected animals in immunoassays designed to detect anti-gE antibodies. In this study a codon optimized synthetic sequence of gE containing highly conserved regions from BoHV-1 and BoHV-5 was expressed in Pichia pastoris. Following expression, the recombinant gE (rgE) was secreted and purified from the culture medium. The rgE was identified by Western blotting (WB) using sera from cattle naturally infected with BoHV-1 and/or BoHV-5, or sera from bovines experimentally infected with wild-type BoHV-5. Sera collected from cattle vaccinated with a BoHV-5 gI/gE/US9¯ marker vaccine failed to recognise rgE. Expression of rgE, based on a sequence containing highly conserved regions from BoHV-1 and BoHV-5, in P. pastoris enabled the production of large quantities of rgE suitable for use in immunoassays for the differentiation vaccinated or infected cattle.
Asunto(s)
Expresión Génica , Herpesvirus Bovino 1/genética , Herpesvirus Bovino 5 , Rinotraqueítis Infecciosa Bovina , Pichia/metabolismo , Proteínas del Envoltorio Viral , Proteínas Virales , Animales , Bovinos , Herpesvirus Bovino 1/metabolismo , Vacunas contra Herpesvirus/farmacología , Rinotraqueítis Infecciosa Bovina/sangre , Rinotraqueítis Infecciosa Bovina/diagnóstico , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificación , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismoRESUMEN
Leptospirosis is a zoonotic disease caused by pathogenic spirochetes from the genus Leptospira, which includes 20 species and more than 300 serovars. Canines are important hosts of pathogenic leptospires and can transmit the pathogen to humans via infected urine. Here, we report the phenotypic and molecular characterization of Leptospira interrogans isolated from Canis familiaris in Southern Brazil. The isolated strain was characterized by variable-number tandem-repeats analysis as L. interrogans, serogroup Icterohaemorrhagiae. In addition, the isolate was recognized by antibodies from human and canine serum samples previously tested by microscopic agglutination test. Ultimately, the expression of membrane-associated antigens (LipL32 and leptospiral immunoglobulin-like proteins) from pathogenic leptospires using monoclonal antibodies was detected by indirect immunofluorescence assay. In conclusion, identification of new strains of Leptospira can help in the diagnosis and control of leptospirosis.
