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Journal of General Virology (2001), 82, 9-15.
© 2001 Society for General Microbiology

Plant

Rapid identification of a tobacco mosaic virus epitope by using a coat protein gene-fragment–pVIII fusion library

Achim Holzem1, Jörg M. Nähring1 and Rainer Fischer1,2

Institut für Biologie I (Botanik/Molekularbiologie), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany1
Fraunhofer Department for Molecular Biotechnology, IUCT, Grafschaft, Auf dem Aberg 1, D-57392 Schmallenberg, Germany2

Author for correspondence: Rainer Fischer (at RWTH Aachen). Fax +49 241 871062. e-mail fischer{at}bio1.rwth-aachen.de

This study describes the identification of the epitope recognized by the tobacco mosaic virus (TMV) coat protein (CP)-specific monoclonal antibody 29 (MAb29) by displaying a CP gene-fragment library on pVIII of filamentous phage M13. More than 80% of the clones isolated after one round of panning bound specifically to MAb29. DNA sequencing of ten randomly chosen MAb29-specific clones and subsequent sequence comparison revealed a common seven amino acid epitope (ELIRGTG) representing amino acids 131–137 of the TMV CP. The reactivity of MAb29 in competition ELISA towards glutathione S-transferase fused to this epitope was stronger than that towards full-length wild-type TMV CP, confirming the epitope sequence determined by gene-fragment phage display. This demonstrated that gene-fragment libraries displayed on the phage surface as fusion proteins with the filamentous bacteriophage gene VIII are useful tools for rapid identification of linear epitopes recognized by MAbs.




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