Abstract：In order to prepare a multi-epitopeantigen of Porcine epidemic diarrheavirus(PEDV) S1 gene chimerized with Norovirus( NoV)P particles. The PEDV S1 multi-cpitope gene sequence was designed and synthesized, enzymatically ligated into the recombinant pGEX-4T-1vector of the NoV P gene,transformed the vector into E.coli DH5a 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 concentrations of Isopropyl-β-D-thiogalactopyranoside (IPTG)at different induction times to determine the optimalinduction 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-partical-PEDV-SE was identified by double digestion and a larget gene band of proximately 510 bp in size was obtained. The molecular mass of the induced recombinant protein was about 79 ku, which was consistent with the expected size. The recombinant protein was expressed in the supernatant after sonication of the fragmented bacterium using 1. 0 mmol/LIPTG at 37 ℃ for 2 h as the optimal induction condition. Both murine-derived GST monoclonal antibody and PEDV-positive scrum specifically recognized the purified 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-SEprotein,which is the basis for further preparation of a multi-epitope oral vaccine against PEDV in pigs.
引用本文:
章程,罗沛,周德荣,付新亮,阳佑天,黄运茂,刘文俊. NoV P颗粒嵌合PEDV S1基因多表位抗原的制备[J]. 仲恺农业工程学院学报, 2024, 37(2): 12-18.
2024, Vol. 37 
