title: Production of an enzymatically active and immunogenic form of ectodomain of Porcine rubulavirus hemagglutinin-neuraminidase in the yeast Pichia pastoris.
abstract: Blue-eye disease (BED) of swine is a viral disease endemic in Mexico. The etiological agent is a paramyxovirus classified as Porcine rubulavirus (PoRV-LPMV), which exhibits in its envelope the hemagglutinin-neuraminidase (HN) glycoprotein, the most immunogenic and a major target for vaccine development. We report in this study the obtaining of ectodomain of PoRV HN (eHN) through the Pichia pastoris expression system. The expression vector (pPICZαB-HN) was integrated by displacement into the yeast chromosome and resulted in a Mut(+) phenotype. Expressed eHN in the P. pastoris X33 strain was recovered from cell-free medium, featuring up to 67 nmol/min/mg after 6 days of expression. eHN was recognized by the serum of infected pigs with strains currently circulating in the Mexican Bajio region. eHN induces antibodies in mice after 28 days of immunization with specific recognition in ELISA test. These antibodies were able to inhibit >80% replication by viral neutralization assays in cell culture. These studies show the obtaining of a protein with similar characteristics to the native HN and which may be a candidate to propose a vaccine or to use the antigen in a serologic diagnostic test.
authors.affiliation: Laboratorio de Bioquímica y Biología Molecular, Centro de Química del Instituto de Ciencias (ICUAP), Edificio 103F, Ciudad Universitaria, Benemérita Universidad Autónoma de Puebla, Puebla, Puebla, Mexico; Posgrado en Ciencias Químicas, Edificio FCQ10, Ciudad Universitaria, Benemérita Universidad Autónoma de Puebla, Puebla, Puebla, Mexico; Laboratorio de Biología Molecular y Virología, Centro de Investigación Biomédica de Oriente, Instituto Mexicano del Seguro Social, Hospital General de Zona No. 5, Km. 4.5 Carretera Federal Atlixco-Metepec, Metepec, Puebla, Mexico.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Cerriteño-Sánchez
authors.affiliation: Laboratorio de Biología Molecular y Virología, Centro de Investigación Biomédica de Oriente, Instituto Mexicano del Seguro Social, Hospital General de Zona No. 5, Km. 4.5 Carretera Federal Atlixco-Metepec, Metepec, Puebla, Mexico.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Santos-López
authors.affiliation: Laboratorio de Bioquímica y Biología Molecular, Centro de Química del Instituto de Ciencias (ICUAP), Edificio 103F, Ciudad Universitaria, Benemérita Universidad Autónoma de Puebla, Puebla, Puebla, Mexico.
authors.forename: Nora Hilda
authors.initials: NH
authors.lastname: Rosas-Murrieta
authors.affiliation: Laboratorio de Biología Molecular y Virología, Centro de Investigación Biomédica de Oriente, Instituto Mexicano del Seguro Social, Hospital General de Zona No. 5, Km. 4.5 Carretera Federal Atlixco-Metepec, Metepec, Puebla, Mexico.
authors.forename: Julio
authors.initials: J
authors.lastname: Reyes-Leyva
authors.affiliation: Laboratorio de Virología, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Km 15.5 Carretera México-Toluca, Palo Alto, Cuajimalpa, Distrito Federal, Mexico.
authors.forename: Sandra
authors.initials: S
authors.lastname: Cuevas-Romero
authors.affiliation: Laboratorio de Bioquímica y Biología Molecular, Centro de Química del Instituto de Ciencias (ICUAP), Edificio 103F, Ciudad Universitaria, Benemérita Universidad Autónoma de Puebla, Puebla, Puebla, Mexico. Electronic address: irma.herrera@correo.buap.mx.
authors.forename: Irma
authors.initials: I
authors.lastname: Herrera-Camacho
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000378:metabolism; D002474:Cell-Free System; D015707:HN Protein / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007114:Immunization; D051379:Mice; D010843:Pichia / Q000235:genetics / Q000254:growth & development*; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019213:Rubulavirus / Q000235:genetics / Q000378:metabolism*; D013552:Swine
chemical_list: D000914:Antibodies, Viral; D015707:HN Protein; D011994:Recombinant Proteins
keywords: Blue eye disease; Hemagglutinin-neuraminidase; PoRV; eHN