Production of an enzymatically active and immunogenic form of ectodomain of Porcine rubulavirus hemagglutinin-neuraminidase in the yeast Pichia pastoris

•A P. pastoris clone producing recombinant hemagglutinin-neuraminidase was obtained.•The produced recombinant protein is enzymatically active.•The purified recombinant protein is immunogenic in a mouse model.•The recombinant protein is recognized by antibodies of PoRV infected pigs. Blue-eye disease...

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Bibliographic Details
Published in:Journal of biotechnology 2016-04, Vol.223, p.52-61
Main Authors: Cerriteño-Sánchez, José Luis, Santos-López, Gerardo, Rosas-Murrieta, Nora Hilda, Reyes-Leyva, Julio, Cuevas-Romero, Sandra, Herrera-Camacho, Irma
Format: Article
Language:English
Subjects:
Animals
Antibodies
Antibodies, Viral - metabolism
Antigens
Biotechnology
Blue eye disease
Cell-Free System
eHN
Hemagglutinin-neuraminidase
HN Protein - chemistry
HN Protein - genetics
HN Protein - immunology
HN Protein - metabolism
Immunization
Mice
Paramyxovirus
Pichia - genetics
Pichia - growth & development
Pichia pastoris
Porcine rubulavirus
PoRV
Protein Domains
Recombinant Proteins - genetics
Recombinant Proteins - immunology
Recombinant Proteins - metabolism
Rubulavirus - genetics
Rubulavirus - metabolism
Strain
Swine
Vaccines
Yeast
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Summary:•A P. pastoris clone producing recombinant hemagglutinin-neuraminidase was obtained.•The produced recombinant protein is enzymatically active.•The purified recombinant protein is immunogenic in a mouse model.•The recombinant protein is recognized by antibodies of PoRV infected pigs. 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 67nmol/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.
ISSN:0168-1656
1873-4863
DOI:10.1016/j.jbiotec.2016.02.035