| Porcine epidemic diarrhea virus?PEDV?is a serious threat to the global swine industry.The difficulty of PEDV proliferation and the lower virus titers by traditional cell culture techinique limits the application of PEDV vaccine.In this study,the recombinant yeast strain anchoring a truncated PEDV S1 protein?COE?on the suface of Saccharomyces cerevisiae was constructed.The expression characteristics of the recombinant strain were evaluted.The immune efficacy of the strain was evaluated as a candidate vaccine by oral routine in mice.The main research results are as follows:1.Immunogenicity analysis and expression domain screening of PEDV S1protein.Through the analysis of the tertiary structure of PEDV S1,antigenicity,epitopes,etc,it is predicted that the 562-567 amino acids of S1 are the key targets for exerting virus neutralization;We chose a truncated sequence about 870bp containing the COE region of S1 for recombinant Saccharomyces cerevisiae construction.2.Construction of recombinant Saccharomyces cerevisiae.The additional recombinant Saccharomyces cerevisiae expression vector was constructed using pYD1 expression plasmid;EBY100 host strain was transformed by Lithium acetate,and additional Saccharomyces cerevisiae recombinants were selected by tryptophan auxotrophic medium;The expression characteristics of the selected recombinants were studied.Western Blot,immunofluorescence and flow cytometry confirmed that the surface of the recombinant Saccharomyces cerevisiae successfully displayed the protein of truncated of S1 containing the key COE region of PEDV.Western Blot results showed that the specific reaction of the target protein with the positive serum of PEDV indicated that the protein had a certain immunogenicity;In addition,the surface-expressed target protein couid be eluted with 100?M DTT;Flow cytometry results showed the percentage of target protein displayed cells was the highest at 36h after induction at 20°C,which was 49.1%.The amount of expressed target protein in the single cell was the highest at 36h after induction at 20°C,which was approximately 24×10-7?g/cfu;3.Immunological characteristics of recombinant Saccharomyces cerevisiae:KM mice were orally administered with recombinant Saccharomyces cerevisiae containing60?g of target protein for 3 times at 10 days interval.After three boost immunization,there was no significant effect on the growth rate and body weight of mice.The spleen index of immunity mice was significantly higher than that of the control group.The neutralizing antibody titer of immunity mice was 1:6 by serum neutralization experiment.The results showed that oral Saccharomyces cerevisiae immunization can stimulate good immune response.In summary,we obtained a recombinant Saccharomyces cerevisiae strain that display the truncated protein containing the key COE region of PEDV S1.The recombinant Saccharomyces cerevisiae can stimulate a good immunogenicity in inoculated mice by oral routine.The recombinant COE Saccharomyces cerevisiae have the advantages of simple use,high safety,and cost less than traditional vaccines,which can be used as a potential vaccine candidate for PEDV control. |
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