Abstract:
【Objective】 The purpose of the study was to prokaryotically express porcine Seneca valley virus (SVV) VP1 protein,to prepare polyclonal antibody against SVV-CH-HB2016 VP1 protein,and to analyze its antigenicity,so as to provide reference for further development of SVV diagnostic kits and subunit vaccines.【Method】 The codon optimization of SVV-CH-HB2016 VP1 gene sequence was carried out, and the plasmid pUCK/VP1-opt was constructed. After double enzyme digestion,pET-28a(+) vector was connected,and the prokaryotic expression plasmid pET-28a-VP1 was constructed. The recombinant plasmid pET-28a-VP1 was transformed into
Escherichia coli BL21(DE3) molecular chaperone receptor cells, and the recombinant VP1 protein was obtained by IPTG induction and expression. The reactivity of VP1 protein was analyzed by the Western blotting method, and purified by denaturation and renaturation of inclusion body. Then,the New Zealand white rabbits were immunized with VP1 protein,and the serum was isolated to obtain the polyclonal antibody of the recombinant VP1 protein. The antibody titer level was determined by indirect enzyme-linked immunosorbent assay(ELISA), and the polyclonal antibody was purified with Protein A+G Agarose antibody purification kit. The specificity of the polyclonal antibody reaction with SVV-CH-HB2016 strain was verified by Western blotting and direct immunofluorescent antibody assay(IFA) methods.【Result】 The prokaryotic expression plasmid pET-28a-VP1 was successfully constructed. The recombinant VP1 protein was successfully expressed and existed mainly in the form of inclusion body under the condition of IPTG final concentration of 0.1 mmol/L and induction at 37℃ for 4 h. The Western blotting analysis result showed that the recombinant VP1 protein could bind specifically to His-Tag Mouse mAb with good reactivity. The recombin antinclusion body protein purified by denaturation and renaturation had high purity and concentration. The titer of polyclonal antibody determined by indirect ELISA was 1:32000. The purified antibody had no obvious heteroband and the purity was more than 90.0%. The results of the Western blotting and IFA identification showed that the polyclonal antibody could specifically recognize SVV-CH-HB2016 strain,and the recombinant VP1 protein had good immunogenicity.【Conclusion】 The recombinant VP1 protein obtained by prokaryotic expression of SVV VP1 protein has good antigenicity, and the prepared polyclonal antibody has good specificity, which can be used as a candidate antigen for the development of diagnostic kits and subunit vaccines.