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Expression and Characterization of HA 1 Protein of Highly Pathogenic H5N1 Avian Influenza Virus for Use in a Serodiagnostic Assay
Author(s) -
Luo L.,
Nishi K.,
MacLeod E.,
Sabara M. I.
Publication year - 2017
Publication title -
transboundary and emerging diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.392
H-Index - 63
eISSN - 1865-1682
pISSN - 1865-1674
DOI - 10.1111/tbed.12382
Subject(s) - biology , recombinant dna , microbiology and biotechnology , virology , ectodomain , hemagglutinin (influenza) , signal peptide , pngase f , antibody , glycosylation , influenza a virus subtype h5n1 , melittin , western blot , influenza a virus , tunicamycin , virus , peptide , biochemistry , gene , immunology , receptor , unfolded protein response
Summary The hemagglutinin ectodomain ( HA 1 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 HA 1 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‐ kD a unglycosylated precursor and a fully processed, mature form of the HA 1 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 ( PNG ase F) further revealed that the recombinant HA 1 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 HA 1. A high yield, 37 μg/ml, of HA 1 protein was obtained from recombinant baculovirus‐infected cell culture supernatant. In addition, the cell surface expression of rHA 1 was detected by indirect immunofluorescent staining and showed biological activity on hemadsorption assays. Recombinant HA 1 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 HA 1‐based ELISA could offer an alternative to other diagnostic approaches for the specific detection of H5 avian influenza virus infection.