Research On Porcine Epidemic Diarrhea Virus (PDEV)S/M Double Genes DNA Vaccine Presented By Attenuated Salmonella

Porcine Epidemic Diarrhea Virus (PEDV) is a swine viral diarrhea with severe vomiting, diarrhea and dehydration symptoms, resulting serious economic losses for the swine industry. The research of PEDV has been focused in the development of stable, secure and efficient vaccine to prevent and control PEDV. In this study, PEDV S gene and M gene are used as target genes to construct a DNA vaccine that was delivered by attenuated Salmonella typhimurium and to carry out immune test on mice to explore its immune efficiency.1. Cloning and prokaryotic expression of PEDV S/M geneThe BL21expression bacteria recombinant prokaryotic expression plasmid pET32a-Sa and pET32a-PMa were constructed after1590-2328bp of S gene and357-596bp of M gene were amplified via PCR acccording to the PEDV CV777sequence in GenBank. The recombinant bacteria respectively expressed fusion proteins of approximate45K Da and28KDa after IPTG induction. The polyclonal antiserum was prepared by immunizing rabbits with these recombinant proteins have good biological activity.2. The construction of recombinant S.typhimurium and studying on its biological characteristicsS gene and M gene were inserted into double-promoter expression plasmid and the eukaryotic expression plasmid pVAXD-PM and pVAXD-PS1-PM were successfully constructed via PCR and enzyme identification. They were transfected into COS-7cells and expression of recombinant plasmids was confirmed by indirect immunofluorscence assay and the transfected COS-7cells displayed specific immunofluorscence, demonstrating that the constructed plasmids pVAXD-PM and pVAXD-PS1-PM could successfully express in vitro. The eukaryotic expression plasmids pVAXD-PM and pVAXD-PS1-PM were transformed by electroporation to construct recombinant attenuated Salmonella typhimurium SL7207(pVAXD-PM) and SL7207(pVAXD-PS1-PM). The growth characteristics and plasmid stability test in vitro indicated that the recombinant bacteria is in the similar growth curve as SL7207and good stability could be observed in the condition without Kan resistance.3. Immunogenicity research of PEDV DNA vaccine on miceThe mice were inoculated orally with recombinant strain at dosages of1×109CFU,1×1010CFU and1×1011CFU per unit of SL7207(pVAXD-PS1)、 SL7207(pVAXD-PM) and SL7207(pVAXD-PS1-PM) respectively for safety analysis, there were no significant difference of appetite, drinking desire and mental condition between oral immunization groups and control group, indicating that the recombinant strain was relatively safe concerning the pathology of Salmonella.Mice of6-week-old were divided into four groups with15in each, they all were orally inoculated at dosages of1×109CFU per unit of mixed recombinant strains SL7207(pVAXD-PS1) and SL7207(pVAXD-PM), and SL7207(pVAXD-PS1-PM) at the dosage of1×109CFU per unit three times, specific PEDV serum IgG and intestinal mucosal IgA antibody were detected by indirect ELISA. The results indicated that the recombinant attenuated Salmonella can provide good immunogenicity by oral immunization, and effectively eliciting production of specified PEDV serum IgG and intestinal IgA antibodies and inducing cellular immune response of mice at the same time.