Expression and purification of S1 protein and preparation of polyclonal antibody for porcine epidemic diarrhea virus
The S1 protein of porcine epidemic diarrhea virus(PEDV)was employed as the research object.The baculovirus system of PEDV S1 was constructed,enabling the stable expression of high-purity S1 protein,and the polyclonal antibody of high-specific S1 protein was prepared.Based on the sequence information of PEDV S1 gene of multiple genotypes,bioinformatics technology was utilized to analyze its physicochemical properties,transmembrane domains,epitopes,and secondary structures.Furthermore,the codon of the S1 gene sequence suitable for the expression of insect cells was optimized,and a Bac-to-Bac baculovirus expression system suitable for the expression of PEDV S1 recombinant protein was constructed through steps such as gene synthesis,vector construction,bacmid transformation,and cell culture.The S1 recombinant protein was purified by nickel column affinity chromatography and ultrafiltration technology,mixed with Freund's adjuvant and emulsified with BALB/c mice,and murine polyclonal antiserum(polyserum)was prepared.Indirect immunofluorescence(IFA),sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and Western Blot(Western Blot)were used to successfully confirm that the S1 recombinant protein could be effectively expressed by the baculovirus expression system established in this study.ELISA and IFA verified the effectiveness of the obtained antibodies in specifically recognizing PEDV-infected Vero cells.In this study,a baculovirus expression system for PEDV S1 was successfully constructed,which stably expressed high-purity S1 protein,and a polyclonal antibody to S1 protein with high specificity was prepared,laying a foundation for the preparation of monoclonal antibodies to porcine epidemic diarrhea and the research and development of detection reagents in the future.
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