Epitope Screening Of The PCV2Cap Protein By Using A Random Peptide-Displayed Library And Polyclonal Antibody

To fully map the epitopes on Cap protein of PCV2, here, a random peptide phage display library was employed to identify epitopes of Cap specific antibodies isolated from pooled sera by affinity chromatography.Firstly, total IgG was isolated from the pooled PCV2positive sera by three consecutive ammonium sulfate precipitations; an affinity column consisting of Cap protein coupled to1ml CNBr-activated sepharose4B was prepared and the Cap-specific antibodies were isolated from the total IgG by chromatography on this Cap column. About0.2mg Cap-specific antibody could be obtained from lml total IgG.Secondly, purified Cap-specific polyclonal antibodies were used as targets to screen a random heptapeptide phage display library, after three rounds of screening, twenty of27randomly selected phage clones had binding activity to Cap-specific antibodies by phage ELISA assays. These were sequenced and when the inserted amino acid sequences were aligned with the Cap protein sequence, eight regions of the Cap protein,53FGYT56,65PSW67,69VDMMR73,79FLPPG84,86SNPRSVPF93,103KVEFWP108,119GSSXXXLDD127and230PLNP233could be deduced. Three of the eight core regions were firsly identified and the others were located on previous studies.According to the phage ELISA results and sequence alignments,8phagetopes represent the eight regions were chose for immunogenicity analysis. The results of Western blot showed all the selected phagetopes could be recognized by the porcine sera. When these phagetopes were put to immunize Kunming mice, two phagetopes53FGYT56and230PLNP233failed to induce a response in mice.The results indicated that two newly mimotopes (86SNPRSVPF93,103KVEFWP108) were identified on Cap protein in our study.