The Research On The Antigenic Characteristics Of FliC From Salmonella Enteritidis In Chickens Using The Technology Of Yeast Surface Display

Salmonella enteritidis(SE) is a non-host-adapted Salmonella. It is not only one of the most seriouspathogens which hinder the development of the poultry industry,but also threats the human healththrough the contaminated poultry products. Flagellin of Salmonella, encoded by the fliC gene, is one ofthe important protective antigens because of its ability to induce a strong immune response in vivo.Studies on dominant antigenic domains of the Fli C protein will contribute to the research on anddevelopment of the salmonella new vaccines, and further reduce the infection of human food-borneS.enteritidisIn this study, the fliC gene of S. enteritidis was randomly digested into 100-500 bp DNA fragmentsin length with DNaseⅠ. The small fragments were inserted into pCTCON-2 vector and transformedinto E.coli to construct a random yeast display library pCTCON-fliC-Lib. The library size was about 2.7x 105 colonies. The genesequencing analysis showed that all fragments in the 50 randomly picked clonesare random in the inserted direction and approximately 100-500 bp in length, and they cover the entirefliC gene.The yeast display library pCTCON-fliC-Lib were transformed into Saccharomyces cerevisiaeEBY-100 strains for expression on the yeast surface.The FliC peptide expressed on the yeast cells werestained with positive sera against S. enteritidis from chickens sorted by FACS for two rounds.Therecombinant plasmids in the sorted yeast clones were extracted and sequenced.The results showed thatthe antigenicity of SM6 Fli C protein is located on the D1(1-201 aa), D2(202-352 aa) and D4(394-505 aa) domains, meanwhile D3(353-393 aa)domain does not detected any visiable antigenicresponses.To further verify the antigenic domains on FliC, the four antigenic domains were expressed on theyeast surface for the analysis of immune sera against SM6 by FACS. At the same time, the fourantigenic domains of FliC were expressed in E. coli and purified to esablish an ELISA assay to analyzethe immune sera against SM6. The results showed that D1 and D2 are dominant antigenic domainscompared to D4 domain. The analyses of chicken immunization and SM6 challenge showed that theresponses against D1, D2, D4 antigenic domains of FliC were able to reduce SM6 colonization inchickens.In summary, this study has successfully displayed the randomly-generated FliC peptides ofS. enteritidis SM6 strain on the yeast surface using yeast surface display technology. It is furtherscreened the dominant antigenic domains of Fli C which are confirmed by FACS and ELISA assays.The work will underlay the theoretical foundation for the development of new vaccines againstS. enteritidis.