Characterization of a monoclonal antibody and its single-chain antibody fragment recognizing the nucleoside Triphosphatase/Helicase domain of the hepatitis C virus nonstructural 3 protein.
We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors. Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371-1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.
Zhang
Z X
ZX
Divisions of Clinical Virology and Basic Oral Sciences, Huddinge University Hospital, S-141 86 Huddinge, Sweden.
Lazdina
U
U
Chen
M
M
Peterson
D L
DL
Sällberg
M
M
D017766:Acid Anhydride Hydrolases / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D002460:Cell Line; D004265:DNA Helicases / Q000276:immunology*; D004274:DNA, Recombinant / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D016174:Hepacivirus / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D043583:Nucleoside-Triphosphatase; D017434:Protein Structure, Tertiary / Q000502:physiology; D017361:Viral Nonstructural Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D004274:DNA, Recombinant; D000939:Epitopes; D007128:Immunoglobulin Fragments; D017361:Viral Nonstructural Proteins; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D004265:DNA Helicases