Construction Of The Recombinant Co-expressing M&N Protein Of Porcine Epidemic Diarrhea Virus In Attenuated Salmonella And The Immune Efficiency Of The Vaccine

Porcine epidemic diarrhea (PED) is induced by porcine epidemic diarrhea virus (PEDV) and characterized with acute watery diarrhea, dehydration, vomiting in young piglets and leads to death with a high mortality rate. It causes important economic losses to the pig produce. With the rapid development of molecular biological technology, it has become a hot point of the research to develop new type gene engineering vaccines, characterized by safety, modifiability, and effectivity. In this study, PEDV M and N protein gene full-length cDNA were amplified by RT-PCR and cloned into pMD18-T Simple vector which were named pMD-M and pMD-N. Sequence analysis showed that two complete genes were consist of681bp、1320bp respectively and were high conservative. pMD-M and pMD-N was double digested respectively using Xba I/Sal I and Xho I IMlu I to identify recombinants. Then the fragments was subcloned into the eukaryotic expression vector pIRES. After identified by enzyme analysis and nucleotide sequencing test, the constructed recombinant plasmid, designated as pIRES-M-N, was transfected into Vero cells. The transient expression protein was detected by indirect immunofluorescence assay (IFA) and Western-blotting. Result showed that pIRES-M-N can be expressed correctly in Vero Cells, and be reactive with polyclonal antibody serum. Then the recombinant plasmid electroporated into S.choleraesuis C500. and the recombinant bacteria was proved to be stabile In vitro. The immunogenicities of this recombinant Attenuated Salmonella constructs were firstly investigated in a mouse mode. Specific anti-PEDV antibodies、 anti-PEDV IgA and IFN-y, IL-4of cytokine, were detected by ELISA. The result showed that the recombinant Attenuated Salmonella could induce higher persistence specific hummune responses in mouse by contrast with naked DNA group after42days (P<0.05), and improve the anti-PEDV IgA level in fecals. It also stimulated Thl/Th2immune response. That provided the clinical basic data and theoretical basis for success of the Attenuated Salmonella vaccine development.