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Selection of scFvs specific for HBV DNA polymerase using ribosome display.

Authors
Lee, MS; Kwon, MH; Kim, KH; Shin, HJ; Park, S; Kim, HI
Citation
Journal of immunological methods, 284(1-2):147-157, 2004
Journal Title
Journal of immunological methods
ISSN
0022-17591872-7905
Abstract
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.
MeSH terms
Amino Acid SequenceAnimalsAntibodies, Viral/bloodAntibody SpecificityBase SequenceChromatography, AffinityDNA-Directed DNA Polymerase/genetics/*immunologyEnzyme-Linked Immunosorbent AssayFemaleHepatitis B virus/*enzymology/geneticsImmunoglobulin Variable Region/genetics/*immunologyMiceMice, Inbred BALB CMolecular Sequence DataProtein Biosynthesis/immunologyRNA, Messenger/chemistry/geneticsReverse Transcriptase Polymerase Chain ReactionRibosomes/immunologySelection, GeneticSequence Alignment
PMID
14736425
Appears in Collections:
Journal Papers > School of Medicine / Graduate School of Medicine > Microbiology
AJOU Authors
권, 명희김, 경민신, 호준박, 선
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