Construction And Immunogenicity Research Of Two Immunodominant Region Of Spike Protein Of Porcine Diarrhea Virus Presented By Attenuated Salmonella Typhimurium

Porcine epidemic diarrhea virus (PEDV) is the causative agent of Porcine epidemic diarrhea (PED) which characterized by vomit, watery diarrhea and severe dehydration, causing high morbidity and mortality in newborn piglets and leading to significant economic losses in swine husbandry. Spike protein is the major inducer of PEDV-neutralizing antibodies. In this study, we used attenuated salmonella typhimurium SL7207 as the carrier for delivery of PEDV spike protein S499-650 domain (aa 499-650) and S499-789 domain (aa 499-789) to structure SL7207(pVAX-S499-650) and SL7207 (pVAX-S499-789). The recombinant S.typhimurium strains SL7207 (pVAX-S499-650) and SL7207 (pVAX-S499-789) as candidate oral mucosal vaccine were evaluated in mice and swine models.1.Construction and identification of eukaryotic expression plasmidsBased on spike gene sequence of PEDV SC-L strain in GenBank, specific primers were designed. With RT-PCR, S499-650 and S499-789 genes were cloned and inserted eukaryotic expression vector pVAXl to structure pVAX-S499-650, pVAX-S499-789. After transfection of COS-7 with pVAX-S499-650、pVAX-S499-789, indirect immunofluorescence assays and western-blotting were performed. The results showed S499-650 and S499-789 were expressed in COS-7 cells and The proteins specifically reacted with rabbit-anti-PEDV IgG which demonstrated the construction of eukaryotic expression plasmids.2.Construction and identification of recombination attenuated S.typhimurium strains harbouring PEDV S genesPlasmids pVAX-S499-650, pVAX-S499-789 were transferred into attenuated S.typhimurium SL7207 separately and SL7207 (pVAX-S499-650),SL7207 (pVAX-S499-789) were constructed. After oral administration of SL7207 (pVAX-S499-650) SL7207 (pVAX-S499-789) at dosage of 1×109 CFU/mouse, The biological characteristics research of SL7207(pVAX-S499-650), SL7207(pVAX-S499-789)showed: SL7207 (pVAX-S499-650), SL7207 (pVAX-S499-789) were steady in vivo; the target genes could be transcribed in the ileum tissue of mouse; recombinant strains could Colonize in liver and spleen of mice and gradually eliminated by host; safety to. BALB/c mouse at the dosage of 1×109 CFU by oral administration. All of this; laying the foundation for evaluating the immunogenicity of recombinant attenuated salmonella strains in piglet.3.Oral immunogenicity of recombination attenuated S.typhimurium strainsFifteen 25-day old piglets were randomly divided into five groups and immunized by oral route with recombination attenuated S.typhimurium SL7207 (PVAX-S499-650), SL7207 (pVAX-S499-789) at the dosage of 1×1012 CFU; intramuscular infected with commercial inactivated vaccine as positive control and SL7207(pVAX), and PBS as negative control, respectively. All the Swine was boosted at two weeks later after primary immunization and serum and mucosal samples were collected at 0,2,4,6 weeks after primary immunization, the levels or neutralization of serum IgG and mucosal IgA, Proliferation of T lymphocytes, Indirect detection of IFN-γ and IL-4 were performed. The significant higher level of neutralizing IgA (P＜0.01) compare to inactivated vaccine demonstrated the advantage of recombination attenuated S.typhimurium strains as oral mucosal vaccine; Proliferative activity of T lymphocytes showed the immunogenicity of recombination attenuated S.typhimurium, high levels of IFN-y and low levels of IL-4 indicating the cellular immune response induced were mainly Thl-like phenotype. Follow-up study will perform at the challenge experiment in newborn piglets.