Rapid identification of a tobacco mosaic virus epitope by using a coat protein gene-fragment-pVIII fusion library.
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.
Holzem
Achim
A
Institut für Biologie I (Botanik/Molekularbiologie), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany1.
Nähring
Jörg M
JM
Institut für Biologie I (Botanik/Molekularbiologie), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany1.
Fischer
Rainer
R
Fraunhofer Department for Molecular Biotechnology, IUCT, Grafschaft, Auf dem Aberg 1, D-57392 Schmallenberg, Germany2.|Institut für Biologie I (Botanik/Molekularbiologie), RWTH Aachen, Worringerweg 1, D-52074 Aachen, Germany1.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002213:Capsid / Q000235:genetics* / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D014027:Tobacco Mosaic Virus / Q000276:immunology*; D014760:Viral Fusion Proteins / Q000235:genetics
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes; D014760:Viral Fusion Proteins