title: Expression and Characterization of HA1 Protein of Highly Pathogenic H5N1 Avian Influenza Virus for Use in a Serodiagnostic Assay.
abstract: The hemagglutinin ectodomain (HA1 subunit) from highly pathogenic avian influenza (HPAI) isolate (A/chicken/Vietnam/14/2005) was cloned and expressed using a baculovirus expression vector. Biosynthesis, glycosylation and secretion of the HA1 proteins, with natural or a melittin signal peptide at the N-terminus and a six-histidine (6xHis) tag at the C-terminus, were examined in insect cells. A 40-kDa unglycosylated precursor and a fully processed, mature form of the HA1 protein migrated around 52 kDa were detected by SDS-PAGE and confirmed by Western blot using H5N1-specific antibody. Treatment of tunicamycin and peptide-N-glycosidase F (PNGase F) further revealed that the recombinant HA1 proteins produced in insect cells were indeed glycosylated with N-linked oligosaccharide side chains. Time-course experiments showed that substitution of the HA natural sequence with the signal sequence from honeybee melittin promoted a high level of expression and efficient secretion of the HA1. A high yield, 37 μg/ml, of HA1 protein was obtained from recombinant baculovirus-infected cell culture supernatant. In addition, the cell surface expression of rHA1 was detected by indirect immunofluorescent staining and showed biological activity on hemadsorption assays. Recombinant HA1 protein-based ELISA was evaluated and appeared to be sensitive and specific for the rapid detection of H5 subtype-specific antibodies in serum samples. No cross-reactivity to antibodies from 15 other influenza A subtypes was detected. Taken together, the newly developed recombinant HA1-based ELISA could offer an alternative to other diagnostic approaches for the specific detection of H5 avian influenza virus infection.
authors.affiliation: National Centre for Foreign Animal Disease, Winnipeg, MB, Canada.
authors.forename: L
authors.initials: L
authors.lastname: Luo
authors.affiliation: National Centre for Foreign Animal Disease, Winnipeg, MB, Canada.
authors.forename: K
authors.initials: K
authors.lastname: Nishi
authors.affiliation: National Centre for Foreign Animal Disease, Winnipeg, MB, Canada.
authors.forename: E
authors.initials: E
authors.lastname: MacLeod
authors.affiliation: National Centre for Foreign Animal Disease, Winnipeg, MB, Canada.
authors.forename: M I
authors.initials: MI
authors.lastname: Sabara
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D016367:Baculoviridae / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000378:metabolism*; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus
keywords: 
  ELISA
  ; baculovirus; hemagglutinin; highly pathogenic avian influenza virus; recombinant protein