Screening And Identification Of An Immunogenic Peptide That Mimics Antigen O9 Of Salmonella By Using A Phage Display Library

Salmonella is a major zoonotic pathogens causing of morbidity and mortality worldwide in both animals and human beings. The lipopolysaccharide (LPS) is one of the major virulent determinants and serotypic antigens of Salmonella. Since polysaccharide is poorly immunogenic due to its T-independent nature, it is necessary to take the conventional and tiresome approach is to synthesize polysaccharide-carrier protein conjugates for inducing T-dependent immune response. A novel alternative way to elicit a T-dependent immune response is by selecting peptides that mimic polysaccharide antigens from a phage display library. We used two phage display libraries consisting of 9-random amino acids inserted in the N-terminal region of the major coat protein pVIII with or without two cysteine flanking the insert of phagemid PC89, named as pVllI-9aa and pVIII-9aa Cys, to screen minotopes by monoclonal antibody (MAb) 3-47-0 which is specific for antigen 09 of Salmonella. After each round of biopanning, the plaque forming units (pfu) and transducing units (TU) of the phage eluates and their amplified samples were titrated. The results of the yields and dot-ELJSA showed that the enrichment of positive signals increased gradually following three rounds of biopanning. 4 The single phage clones were amplified from the third round eluate, and then the specific clones were selected by using dot-ELISA. Two positive clones designated as gm68 and gm62 were identified and amino acid sequence YQKWYLPKS and SIIIIVRGGGG of two oligopeptides, respectively, were deduced after DNA sequencing. The phage clone gm68 blocked the binding of MAb 3-47-0 to antigen 09 of Salmonella enteritidis, and the inhibition rate was higher than 65% by 1010 TU phages in cELISA, while the inhibition rate of gm62 was only 20%. This result showed that peptide YQKWYLPKS displayed by gm68 mimicked polysaccharide of antigen 09, and was better than that of gm62. In order to detennine immunogencity of the selected mimicking peptides, ICR mice were immunized subcutaneously with phage gm68, gm62 and M13K07, respectively, and their sera were tested by indirect immunofluorescent assay (IFA) and competitive ELISA. The results demonstrated showed that gm68 can elicit a substantially high level of antibodies against antigen 09, which can bind to Salmonella spp. of serogroup D including S. enteritidis, S. gallinarum, but cannot react with Salmonella strains of other serogroups, including S. kiel, S. typhimurium, S. bonn, S. give, S. illinois, S. aberdeen, S. urbana. In contrast, the phage clone gm62 and Ml 3K07 did not induce specific antibodies against antigen 09. These results revealed that peptide YQKWYLPKS was an immunogenic minotope of antigen 09 with high specificity. We reported here for the first time an immunogenic peptide YQKWYLPKS mimicking of Salmonella polysaccharides. It would be very useful for the study pathogenesis and immune protection of Salmonella antigen 09 and for developing anti-carbohydrate peptide vaccines or DNA vaccines.