A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) librarieson the surface of filamentous phage. This vector contains two long DNA “stuffer” fragments for easier differentiationof the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acidphosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified formof the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III andlight chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide,whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was usedto generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragmentantibody directed against the nucleoprotein (NP) of Influenza A virus.

A phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments

MANCINI, NICASIO
Secondo
;
CLEMENTI, NICOLA;CLEMENTI, MASSIMO
Penultimo
;
BURIONI, ROBERTO
Ultimo
2012-01-01

Abstract

A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) librarieson the surface of filamentous phage. This vector contains two long DNA “stuffer” fragments for easier differentiationof the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acidphosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified formof the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III andlight chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide,whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was usedto generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragmentantibody directed against the nucleoprotein (NP) of Influenza A virus.
2012
Phagemid vector, Combinatorial antibody library, Phage display, Human monoclonal antibody fragments
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11768/47183
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