Selection of immunodominant mimics of IROMP-99 of rabbit Pasteurella multocida from a random 12-peptide library.
A random 12-peptide library was used to screen immunodominant mimics of 99kDa iron-regulated outer membrane protein (IROMP-99) of rabbit Pasteurella multocida. In the present study, expression of IROMPs of rabbit P. multocida strain C51-12 were analyzed by SDS-PAGE, and Western blot to determine the specificity of rat antiserum antibodies against IROMP-99. Only IROMP-99 whose expression was induced under iron-restricted conditions was detected on nitrocellulose paper. The phage display library was screened with rat normal and IROMP-99-specific antiserum. The positive phage clones were identified using enzyme-linked immunoadsorbent assay (ELISA) and inhibition assays for their reactivity to the antiserum. Out of the 18 randomly selected positive clones that showed higher reactivity to rat antiserum, only ten clones efficaciously inhibited binding of rat antisera to IROMPs and their displayed peptides were determined. Alignment using DNAStar-MegAlign software, results showed that motif WHxTxP was highly conserved among nine clones, only clone A7 had no obvious linear homology with either. Our findings suggest that the motif WHxTxP could be an immunodominant mimic epitope of IROMP-99 of rabbit P. multocida strain C51-12.
Key Lab Animal Disease Diagnostic & Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, PR China.
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D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology*; D001428:Bacterial Vaccines / Q000276:immunology; D015153:Blotting, Western / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D016056:Immunodominant Epitopes / Q000235:genetics / Q000276:immunology; D007501:Iron / Q000378:metabolism; D033862:Iron-Binding Proteins; D008970:Molecular Weight; D016979:Pasteurella multocida / Q000276:immunology*; D019151:Peptide Library; D033902:Periplasmic Binding Proteins; D011817:Rabbits; D051381:Rats; D012641:Selection, Genetic; D016415:Sequence Alignment / Q000662:veterinary; D017386:Sequence Homology, Amino Acid; D013047:Specific Pathogen-Free Organisms
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D016056:Immunodominant Epitopes; D033862:Iron-Binding Proteins; D019151:Peptide Library; D033902:Periplasmic Binding Proteins; D007501:Iron