Cholera toxin B subunit(CTB)can enhance antigen presentation and promote T cell pro-liferation,B cell differentiation and B cell isotype conversion.Moreover,woodchuck hepatitis virus post transcriptional regulatory element(WPRE)can enhance gene expression efficiency by optimi-zing RNA polyadenylation,denuclearization and/or translation.In order to construct porcine epi-demic diarrhea virus like particles(VLPs)chimerized with CTB and WPRE and evaluate their im-munogenicity,the G Ⅱ type PEDV S gene,combined with the elements promoting the protein ex-pression and enhancing immune effects,was synthesized by the company and cloned into pET32a(+).Af-ter double enzyme digestion and gel recovery,the gene named as TSCW was cloned into pFastBacl to construct the recombinant plasmid pFastBac-TSCW.pFastBac-TSCW was further transformed into DH10Bac competent cells to obtain recombinant bacmid Bacmid-TSCW.Subsequently,the Bacmid-TSCW was transfected into sf9 cells to obtain recombinant baculovirus BV-TSCW.After co-infection of BV-TSCW and BV-M into sf9 cells,viral like particles VLP-TSCW was obtained and used to immunize mice to evaluate its immunogenicity.The results showed that the recombi-nant plasmid pFastBac-TSCW and bacmid Bacmid-TSCW were successfully constructed.After transfection of sf9 cells with recombinant baculovirus,significant cytopathic effects were observed.PCR and Western blot results showed that the recombinant baculoviruses existed stably in sf9 cells and the target proteins was also expressed stably.In addition,the electron microscopy results showed that BV-TSCW and BV-M successfully assembled into viral like particles VLP-TSCW.Furthermore,ELISA results indicated that VLP-TSCW induced high level specific antibodies.The above results laid the foundation for further optimization,design and development of PEDV VLPs subunit vaccines.

Cholera toxin B subunit(CTB)can enhance antigen presentation and promote T cell pro-liferation,B cell differentiation and B cell isotype conversion.Moreover,woodchuck hepatitis virus post transcriptional regulatory element(WPRE)can enhance gene expression efficiency by optimi-zing RNA polyadenylation,denuclearization and/or translation.In order to construct porcine epi-demic diarrhea virus like particles(VLPs)chimerized with CTB and WPRE and evaluate their im-munogenicity,the G Ⅱ type PEDV S gene,combined with the elements promoting the protein ex-pression and enhancing immune effects,was synthesized by the company and cloned into pET32a(+).Af-ter double enzyme digestion and gel recovery,the gene named as TSCW was cloned into pFastBacl to construct the recombinant plasmid pFastBac-TSCW.pFastBac-TSCW was further transformed into DH10Bac competent cells to obtain recombinant bacmid Bacmid-TSCW.Subsequently,the Bacmid-TSCW was transfected into sf9 cells to obtain recombinant baculovirus BV-TSCW.After co-infection of BV-TSCW and BV-M into sf9 cells,viral like particles VLP-TSCW was obtained and used to immunize mice to evaluate its immunogenicity.The results showed that the recombi-nant plasmid pFastBac-TSCW and bacmid Bacmid-TSCW were successfully constructed.After transfection of sf9 cells with recombinant baculovirus,significant cytopathic effects were observed.PCR and Western blot results showed that the recombinant baculoviruses existed stably in sf9 cells and the target proteins was also expressed stably.In addition,the electron microscopy results showed that BV-TSCW and BV-M successfully assembled into viral like particles VLP-TSCW.Furthermore,ELISA results indicated that VLP-TSCW induced high level specific antibodies.The above results laid the foundation for further optimization,design and development of PEDV VLPs subunit vaccines.