Expression And Mucosal Immunogenicity Of Recombinant PDEV S1 Protein With BmNPV

Porcine epidemic diarrhea(PED)is caused by porcine epidemic diarrhea virus(PEDV),which has a very high morbidity and mortality of piglets.It is urgent to develop new safe and effective vaccines.The S1(1~735 aa)domain of PEDV spike protein S is located on the envelope of the virus,and contains the major neutralizing epitope and receptor binding domain of the virus,which is closely related to the antigenicity and the invasion of the virus.In this paper,S1 was inserted into the GP64 signal peptide and specific membrane anchoring structure(TMD)using the Bac-to-Bac system and fused with the eGFP gene to construct a transfer vector pFastBacHTB-SP-eGFP-TMD.The recombinant BmBacmid-SP-S1-eGFP-TMD was successfully obtained by transforming BmBacmid/DH10 Bac competent cells with pFastBacHTB-SP-eGFP-TMD.After transfection of BmN cells with BmBacmid-SP-S1-eGFPTMD,Western Blot analysis detected that the fusion protein was detected with the presence of two bands of about 150 kDa and 400 kDa respectively,and we preliminary speculated that the produced S1 protein was N-glycosylated and the S1 trimer formed.The recombinant virus was aerosol inhalated by BALB/c mice,with using WT-BmNPV as a negative control and TGEV/PEDV inactivated vaccine as a positive control.Indirect ELISA detection of PEDV specific antibody in serum of mice was conducted and the results showed that higher levels of PEDV-specific antibodies were detected in rvBmBacmid-SP-S1-eGFP-TMD and TGEV/PEDV inactivated vaccine,and the titer was up to 1: 6400,indicating that rvBmBacmidSP-S1-eGFP-TMD and TGEV/PEDV inactivated vaccine can be a good cause of humoral immune response in mice.ELISA detection of mice serum and bronchoalveolar lavage fluid(BALF)secretion IgA(sIgA)showed that the concentration of sIgA in rvBmBacmid-SPS1-eGFP-TMD immunized group was significantly higher than that in TGEV/PEDV inactivated vaccine immunization group,indicating that rvBmBacmid-SP-S1-eGFP-TMD immunized mice can stimulates mice to produce high levels of sIgA,causing a strong mucosal immune response.Splenic lymphocyte proliferation experiment showed that compared with the WT group,rvBmBacmid-SP-S1-eGFP-TMD immunized group and TGEV/PEDV inactivated vaccine immunized group were significantly proliferated(P <0.05),indicating that when these two groupes re-exposure to antigens will perform a proliferative effect and have a significant stimulating effect on mouse lymphocyte activation.The percentage of CD8+T cells in rvBmBacmid-SP-S1-eGFP-TMD group and TGEV/PEDV inactivated vaccine group was higher than that in WT group,indicating that these two groupes can make mouse lymphocytes a good differentiation ability,with good ability to activate T cells to produce immune response.In summary,the recombinant silkworm baculovirus that we prepared for immunized mice has made a preliminary effect,indicating that the recombinant virus can be used as a candidate vaccine of PEDV mucosal vaccine for further development and research.