Production of a truncated recombinant HA1 for influenza A H9 subtype screening.
Hemagglutinin is the major component of membrane protein and plays a major role in virus entry into host cells through their receptors and it is predicted to elicit the production neutralizing antibodies. Our aim is to assess the potential of a truncated rHA1 domain, encoding residues 157-260 to detect influenza A H9 specific antibodies. The predicted characteristics of this protein revealed that it is a hydrophobic protein possessing predominant antigenicity and composed of random coils (48%) and extended strand (28%) but few α-helix (6.33%) and β-sheet (7%). A 312 pb HA1 gene was amplified and cloned in pET23b(+) vector including an C-terminal polyHis as a fusion partner, transformed and expressed in Escherichia coli cells as inclusion bodies. The truncated protein was solubilized with 8 M urea, purified by immobilized metal affinity chromatography and then detected by western blot with anti-His and H9-specific polyclonal antibodies. The test demonstrated high specificity (100%) and sensibility (98%). The immunoreactivity of the truncated rHA1 assessed revealed that only antisera against H9 yielded a specific and strong reactivity, with no cross-reactivity against negative sera. This study demonstrates that the truncated rHA1 may serve as a useful tool for rapid and easy surveillance of H9 infection.
University Tunis El Manar, Institute Pasteur of Tunis, Laboratory of Epidemiology and Veterinary Microbiology, 13, Place Pasteur, BP 74, Tunis-Belvédère, 1002, Tunisia. Electronic address: wafa.tombari@istmt.utm.tn.
Wafa
W
Tombari
University Tunis El Manar, Institute Pasteur of Tunis, Laboratory of Epidemiology and Veterinary Microbiology, 13, Place Pasteur, BP 74, Tunis-Belvédère, 1002, Tunisia.
Abdeljelil
A
Ghram
D000818:Animals; D000914:Antibodies, Viral / Q000737:chemistry / Q000276:immunology*; D002645:Chickens; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry / Q000276:immunology*; D053127:Influenza A Virus, H9N2 Subtype / Q000276:immunology*; D005585:Influenza in Birds / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011994:Recombinant Proteins; C493831:hemagglutinin HA1, influenza B virus
Antigenicity; H9 subtype; HA1 unit; Prokaryotic system; Recombinant protein; Structure prediction