Preparation of NoV P-particle chimeric PEDV S1 gene multi-epitope antigen
In order to prepare a multi-epitope antigen of Porcine epidemic diarrhea virus(PEDV)S1 gene chimerized with Norovirus(NoV)P particles.The PEDV S1 multi-epitope gene sequence was de-signed and synthesized,enzymatically ligated into the recombinant pGEX-4T-1 vector of the NoV P gene,transformed the vector into E.coli DH5α receptor cells,and extracted the plasmid for double digestion verification and sequencing.The correctly sequenced recombinant plasmids were transformed into BL21-expressing organisms,and the recombinant proteins were analyzed for solubility using different concentra-tions of Isopropyl-β-D-thiogalactopyranoside(IPTG)at different induction times to determine the optimal induction conditions.Western blot detection of purified recombinant proteins using murine Glutathione-S-transferase(GST)monoclonal antibody and PEDV-positive serum.The recombinant plasmid NoV P-par-tical-PEDV-SE was identified by double digestion and a target gene band of approximately 510 bp in size was obtained.The molecular mass of the induced recombinant protein was about 79 ku,which was con-sistent with the expected size.The recombinant protein was expressed in the supernatant after sonication of the fragmented bacterium using 1.0 mmol/L IPTG at 37 ℃ for 2 h as the optimal induction condition.Both murine-derived GST monoclonal antibody and PEDV-positive serum specifically recognized the puri-fied recombinant protein,indicating that the recombinant protein had good reactogenicity.In this study,a large amount of recombinant protein with high purity was successfully obtained by in situ expression and purification of NoV P-partical-PEDV-SE protein,which is the basis for further preparation of a multi-epitope oral vaccine against PEDV in pigs.
Nov P-particalporcine epidemic diarrhea virusprotein immunogenicityvaccine
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