Hepatitis C epitopes from phage-displayed cDNA libraries and improved diagnosis with a chimeric antigen.
A novel method for cloning DNase I fragments into bacteriophage display vector fUSE2 was used to create libraries expressing hepatitis C virus (HCV) protein fragments on the phage surface. Selection by panning with a mixture of sera from five HCV-seropositive individuals enabled identification of antigenic determinants in NS3 (amino acids 1,383-1,415), NS4 (amino acids 1, 930-1,938), and NS5 (amino acids 2,088-2,104). The NS3 result is the most accurate location to date of a major conformational determinant that cannot be mimicked by short peptides. Any expressed sequence from the phage library can be excised with Bgl II and cloned directly into the Bgl II site of an appropriate plasmid for bacterial expression. This enables production of chimeric proteins containing multiple antigenic determinants, illustrated by co-expression of the NS4P (amino acids 1,930-1,938) epitope with an NS4N fragment (amino acids 1,644-1,812) containing at least three linear HCV epitopes. When used to screen 35 individual HCV-positive sera by enzyme-linked immunosorbent assay (ELISA), the chimeric antigen detected eight more positives than NS4N alone and gave increased immunoreactivity with others. This approach of identifying antigenic regions by phage display and then co-expressing them as chimeric proteins may be generally applicable to the production of improved diagnostic antigens and recombinant vaccines.
Pereboeva
L A
LA
MRIC Biochemistry Group, N. E. Wales Institute, Wrexham, England.
Pereboev
A V
AV
Wang
L F
LF
Morris
G E
GE
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes; D015723:Gene Library; D016174:Hepacivirus / Q000235:genetics / Q000276:immunology*; D006526:Hepatitis C / Q000175:diagnosis* / Q000276:immunology; D018936:Hepatitis C Antigens / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D017386:Sequence Homology, Amino Acid; D017361:Viral Nonstructural Proteins / Q000235:genetics
D000939:Epitopes; D018936:Hepatitis C Antigens; C093542:NS4 protein, hepatitis C virus; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins