Rotavirus VP7 epitope mapping using fragments of VP7 displayed on phages

cDNA copies of the complete porcine rotavirus CRW-8 VP7 gene were randomly digested to fragments of about 30-60 or 30-500 base pairs by DNase1 in the presence of Mn2+. The fragments were cloned and expressed in a filamentous phage fd-tet-derived vector to create specific-gene-related peptide librari es. Polyclonal antibodies were then used to pan the SGRP libraries for anti body-binding phages. Analysis of the phage isolates revealed that the major ity (86%) of them only had a single insert. However, phages displaying comp osite inserts containing the VP7 antigenic regions A, B, and C, originally defined by neutralising monoclonal antibody escape mutants, were also isola ted. Inserts containing A or C region peptide were found to contain extra s equences from the C region, while the B region epitope was linear and had a dditional sequence from either upstream or downstream. In addition a domina nt and possibly non-neutralising VP7 epitope was identified around amino ac ids 263-270. One of the recreated antigenic epitopes has also been fused to the outer membrane protein A (OmpA) of Escherichia coli and shown to maint ain its antigenicity. The results in this study may have significant implic ation for recreation of conformational epitopes and vaccine development. (C ) 2000 Elsevier Science Ltd. All rights reserved.