Amino-terminal epitopes are exposed when full-length open reading frame 2 of hepatitis E virus is expressed in Escherichia coli, but carboxy-terminal epitopes are masked.
We constructed a panel of overlapping and non-overlapping fragments of cDNA derived from open reading frame 2 (ORF2) of hepatitis E virus (HEV) and fused to the gene encoding glutathione S-transferase (GST), from which proteins were expressed in Escherichia coli. IgG-specific immunoreactivity against each protein was measured by Western immunoblotting using sera from experimentally infected Rhesus macaques (Macaca mulatta) or from HEV-infected patients. Under these conditions, full-length ORF2 protein (GST-ORF2) was strongly reactive with acute-phase sera from either macaques or patients, but was poorly reactive with convalescent sera. Recombinant protein GST-ORF2.3, representing amino acids 1-110 of the 660 encoded by ORF2, demonstrated a pattern of reactivity largely indistinguishable from the full-length protein. Conversely, GST-ORF2.1, representing amino acids 394-660 of the ORF2 protein was strongly reactive with both acute- and convalescent-phase sera. Extension of GST-ORF2.1 towards the N-terminus led to a progressive loss of convalescent-phase reactivity, apparent with as few as 20 additional HEV-specific amino acids. Deletion of 40 or more amino acids from the N-terminus of ORF2.1 also led to reduced convalescent-phase reactivity, however a protein representing this "reactive" region, containing amino acids 394-473, was poorly reactive, suggesting that the convalescent-reactive epitopes are conformational. Expression of full-length ORF2 protein in E. coli therefore masks the convalescent-reactive epitopes within the C-terminal part of the protein, without affecting N-terminal, acute-reactive epitopes.
Li
F
F
Macfarlane Burnet Centre for Medical Research, Fairfield, Melbourne, Australia.
Torresi
J
J
Locarnini
S A
SA
Zhuang
H
H
Zhu
W
W
Guo
X
X
Anderson
D A
DA
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000235:genetics / Q000276:immunology; D016751:Hepatitis E / Q000097:blood / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D008253:Macaca mulatta; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D000914:Antibodies, Viral; D000956:Antigens, Viral; D000939:Epitopes; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase