Selection of scFvs specific for HBV DNA polymerase using ribosome display.
We applied a ribosome display technique to a mouse scFv library to select single chain variable fragments (scFvs) specific for the terminal protein (TP) of hepatitis B virus (HBV) DNA polymerase. Synthetic TP-peptide was used as an antigen to obtain scFvs that recognize specific epitopes within the TP domain, the priming site of HBV DNA polymerase. The scFv DNA library was transcribed in vitro to mRNA for ribosome display. scFv-ribosome-mRNA complexes were produced using a rabbit reticulocyte lysate system, and were panned against TP-peptide under appropriate conditions. After each panning, putative scFv-encoding genes were recovered by RT-PCR, and the analysis showed that scFv-ribosome-mRNA complexes were specifically selected by the TP-peptide. We used a radioimmunoassay to show that the TP-peptide-specific scFv pools were enriched through the selection process. Selected scFvs showed binding activity for both the TP-peptide and the HBV DNA polymerase protein in an ELISA. Sequence analysis showed that each TP-specific scFv had a different sequence, and that random mutagenesis was mediated by ribosome display.
Lee
Myung-Shin
MS
Department of Microbiology, Ajou University School of Medicine, Woncheon-dong 5, 442-749, Suwon, South Korea.
Kwon
Myung-Hee
MH
Kim
Kyongmin Hwang
KH
Shin
Ho-Joon
HJ
Park
Sun
S
Kim
Hyung-Il
HI
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D000918:Antibody Specificity; D001483:Base Sequence; D002846:Chromatography, Affinity; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006515:Hepatitis B virus / Q000201:enzymology* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000276:immunology; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000276:immunology; D012641:Selection, Genetic; D016415:Sequence Alignment
D000914:Antibodies, Viral; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D004259:DNA-Directed DNA Polymerase