tetano
Editor, Senior Moderator
Transbound Emerg Dis. 2015 Jun 4. doi: 10.1111/tbed.12382. [Epub ahead of print]
[h=1]Expression and Characterization of HA1 Protein of Highly Pathogenic H5N1 Avian Influenza Virus for Use in a Serodiagnostic Assay.[/h] Luo L[SUP]1[/SUP], Nishi K[SUP]1[/SUP], MacLeod E[SUP]1[/SUP], Sabara MI[SUP]1[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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.
? Her Majesty the Queen in Right of Canada 2015 Reproduced with the permission of the Canadian Food Inspection Agency.
[h=4]KEYWORDS:[/h] ELISA ; baculovirus; hemagglutinin; highly pathogenic avian influenza virus; recombinant protein
PMID: 26040437 [PubMed - as supplied by publisher]
[h=1]Expression and Characterization of HA1 Protein of Highly Pathogenic H5N1 Avian Influenza Virus for Use in a Serodiagnostic Assay.[/h] Luo L[SUP]1[/SUP], Nishi K[SUP]1[/SUP], MacLeod E[SUP]1[/SUP], Sabara MI[SUP]1[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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.
? Her Majesty the Queen in Right of Canada 2015 Reproduced with the permission of the Canadian Food Inspection Agency.
[h=4]KEYWORDS:[/h] ELISA ; baculovirus; hemagglutinin; highly pathogenic avian influenza virus; recombinant protein
PMID: 26040437 [PubMed - as supplied by publisher]