A HUMAN-ANTIBODY SPECIFIC FOR HEPATITIS-C VIRUS CORE PROTEIN - SYNTHESIS IN A BACTERIAL SYSTEM AND CHARACTERIZATION

Citation
G. Esposito et al., A HUMAN-ANTIBODY SPECIFIC FOR HEPATITIS-C VIRUS CORE PROTEIN - SYNTHESIS IN A BACTERIAL SYSTEM AND CHARACTERIZATION, Gene, 164(2), 1995, pp. 203-209
Citations number
44
Categorie Soggetti
Genetics & Heredity
Journal title
GeneACNP
ISSN journal
03781119
Volume
164
Issue
2
Year of publication
1995
Pages
203 - 209
Database
ISI
SICI code
0378-1119(1995)164:2<203:AHSFHV>2.0.ZU;2-I
Abstract
The cDNA coding for the Fab fragment of the human B12.F8 antibody (Ab) , directed against the putative nucleocapsid component (core protein) of hepatitis C virus (HCV), was cloned in the prokaryotic phagemid vec tor, pHEN-1, to obtain its expression in Escherichia coli. The functio nality and specificity of the recombinant Ab, called B12Fab, were exam ined by Western blot and ELISA using recombinant HCV core protein as a ntigen. The specificity of B12Fab was further confirmed by ELISA with the 33-mer peptide epitope recognized by the original whole B12.F8 Ab. By immunofluorescence, the recombinant B12Fab was shown to recognize HCV core protein produced in cells transfected with HCV cDNA, indicati ng that the recombinant B12Fab is suitable as a diagnostic tool for ti ssue localization of the virus. The B12Fab also functioned when displa yed on phage particles, providing the basis for future experiments of in vitro affinity maturation and selection of mutants. The variable ch ain coding regions of the recombinant B12Fab clone were sequenced and the V-gene usage was determined by comparison with the V(k)appa and V- H germline sequences. The B12Fab V-kappa chain belongs to the subgroup II and shows the highest degree of homology with the A(3) germline ge ne, whereas the sequence of the V-H chain is strictly related to that of the Humhv3019b18 gene of the V(H)3 family. These results are, to ou r knowledge, the first report of molecular cloning and characterizatio n of a functional human Ab specific for an HCV antigen.