Prokaryotic Expression of N Protein of Porcine Epidemic Diarrhea Virus and Preparation of Polyclonal Antibodies
[Objective]The purpose of this study was to express the N protein of Porcine epidemic diarrhea virus(PEDV)using a prokaryotic expression system,and prepare polyclonal antibody with high efficiency and specificity.[Method]Bioinformatics tools were used to analyze the amino acid sequence of PEDV N protein and predict its antigenicity.Recombinant N protein was induced by prokaryotic expression vector pCold Ⅰ,and the recombinant protein was identified by SDS-PAGE and Western blotting.New Zealand White rabbits were immunized with the preparation adjuvant of rabbit polyclonal antibody mixed with purified recombinant N protein,and serum was collected to prepare polyclonal antibody.The titer of recombinant N protein polyclonal antibody was determined by indirect ELISA,and the polyclonal antibody was verified by Western blotting and indirect immunofluorescence assay(IFA).[Result]The results of bioinformatics prediction show that N protein didn't contain signal peptide and transmembrane structure,and had good antigenicity and solubility.SDS-PAGE results showed that the recombinant N protein approximately 58 ku in size,existed primarily in a soluble form.Western blotting revealed that the protein specifically react with anti-His labeled mouse monoclonal antibodies.Indirect ELISA results demonstrated that the titer of polyclonal antibody could reach 1∶204 800.Western blotting results showed that recombinant N protein and N protein in PEDV infected Vero cell samples could be specifically identified when the antibody dilution was 1∶5 000.IFA results showed that when the dilution was 1∶1 000,the antibody could effectively identify N protein in PEDV-infected cell samples.[Conclusion]The rabbit anti-N protein polyclonal antibody with high efficiency and good specificity was successfully prepared,providing experimental materials for further study of PEDV N protein function,understanding PEDV replication mechanism and virus-host cell interaction,and laying a foundation for the development of PEDV diagnosis and detection methods.
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