Journal
JOURNAL OF IMMUNOLOGICAL METHODS
Volume 284, Issue 1-2, Pages 147-157Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jim.2003.10.009
Keywords
HBV DNA polymerase; ribosome display; scFv; terminal protein
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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. (C) 2003 Elsevier B.V.. All rights reserved.
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