A human antibody specific for hepatitis C virus core protein: synthesis in a bacterial system and characterization.
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 vector, pHEN-1, to obtain its expression in Escherichia coli. The functionality and specificity of the recombinant Ab, called B12Fab, were examined by Western blot and ELISA using recombinant HCV core protein as antigen. 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, indicating that the recombinant B12Fab is suitable as a diagnostic tool for tissue localization of the virus. The B12Fab also functioned when displayed on phage particles, providing the basis for future experiments of in vitro affinity maturation and selection of mutants. The variable chain coding regions of the recombinant B12Fab clone were sequenced and the V-gene usage was determined by comparison with the V kappa and VH germline sequences. The B12Fab V kappa chain belongs to the subgroup II and shows the highest degree of homology with the A3 germline gene, whereas the sequence of the VH chain is strictly related to that of the Humhv3019b18 gene of the VH3 family. These results are, to our knowledge, the first report of molecular cloning and characterization of a functional human Ab specific for an HCV antigen.
Esposito
G
G
Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Pomezia (Roma), Italy.
Scarselli
E
E
Cerino
A
A
Mondelli
M U
MU
La Monica
N
N
Traboni
C
C
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004926:Escherichia coli; D006367:HeLa Cells; D016174:Hepacivirus / Q000276:immunology*; D018937:Hepatitis C Antibodies / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D014162:Transfection; D014758:Viral Core Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018937:Hepatitis C Antibodies; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D014758:Viral Core Proteins