Preparation And Immunogenicity Analysis Of Porcine Epidemic Diarrhea Virus COE Subunit Vaccine

Porcine epidemic diarrhea(PED)is an acute,highly infectious and fatal intestinal disease caused by the porcine epidemic diarrhea virus(PEDV).Since the first discovery of PED in Britain in the 1970 s,the disease has spread all over the world,causing irreparable economic losses to the global breeding industry and farmers and herdsmen.Since the beginning of this century,with the successful development and wide application of the PEDV vaccine,the effect of prevention and treatment of PEDV has been remarkable.In 2010,the epidemic of PED occurred in many places in China.The vaccine based on the classical strains CV777 and DR13 could not protect piglets from infection.It suggested that the outbreak of PED might be caused by the evolution and variation of PEDV into different genotypes.Therefore,for the diversified strains of PEDV,the traditional vaccine could not protect piglets from infection.At present,there are many vaccines against PEDV in the market.These vaccines,which are generally inoculated by parenteral route,can not effectively induce high titers of specific serum IgG antibody and mucosal SIgA antibody,resulting in poor mucosal protection against PEDV infection.At present,there is still a lack of PEDV vaccine and immune strategy that can provide adequate mucosal protection in the intestine.Object:(1)To analyze the reasons why the current PEDV vaccine in the market is not enough to provide effective protection;(2)To analyze and confirm that it can be used as antigen gene for PEDV subunit vaccine,and to explore the induced expression conditions and purification methods of the recombinant protein through prokaryotic expression system,so as to lay the foundation for the preparation of PEDV subunit vaccine;(3)To evaluate the immunogenicity of PEDV subunit vaccine,preliminarily study the immunization strategy of PEDV COE subunit vaccine,and explore its potential value in the development of PEDV related vaccine.Methods:(1)Analyze the homology of the COE(499 aa ～ 638 aa)amino acid sequence of domestic PEDV epidemic strains since 2010,and obtain the COE gene sequence of the relatively conservative strains through the whole gene synthesis method;(2)Use bioinformatics tools to analyze the basic characteristics,secondary structure,and epitope information of COE genes;(3)Use p ET32a(+)prokaryotic expression system to express and purify recombinant COE protein;(4)Immunize piglets with the purified recombinant COE protein,collect serum and mucosal samples for each time period,establish an indirect ELISA method for detecting PEDV specific antibodies with recombinant COE protein antigen,and detect specific IgG antibodies,feces and saliva in the serum before and after immunization Changes in the levels of specific SIgA antibodies and cytokines in mucosal tissues were used to comprehensively evaluate the immunogenicity of porcine epidemic diarrhea virus COE subunit vaccines through humoral and cellular immunity.Results:(1)Analyzing and comparing the homology of the COE amino acid sequence of domestic PEDV epidemic strains in recent years,selecting the relatively conservative strain CH/PDS/2015(Gen Bank accession number: KU237224,Henan,China)as the template,and successfully synthesizing the COE gene.Bioinformatics analysis results show that the intercepted COE protein(499aa～638 aa)does not have signal peptide and transmembrane domain,and the spatial structure is relatively tight.B cell epitope prediction results show that COE protein contains abundant B cell epitopes and is expected to become a candidate target for subunit vaccines.(2)Construct pcDNA3.1(+)-COE eukaryotic recombinant plasmid,and detect the successful expression at the gene level of 293 T cells.The recombinant prokaryotic expression strain pET-32a-COE-Trans B(DE3)was constructed.The recombinant COE protein with a size of about 35 k Da was expressed in the form of inclusion bodies,and the purification effect was good.The preparation method of PEDV subunit vaccine was successfully constructed.(3)Recombinant COE protein and CpG-ODN+Curdlan can stimulate the specific IgG in piglet serum to increase significantly(p<0.001)through intramuscular injection and maintain a high level for a long time.After oral immunization,low-level and transient specific SIgA antibodies were detected in stool and saliva samples.Conclusion: PEDV COE subunit vaccine has good immunogenicity and can stimulate the body to produce high levels of specific antibodies;The new immunization strategy of intramuscular injection(primary immunization)and oral administration(enhanced immunization)of antigen combined with CpG ODN + curdlan laid a theoretical foundation for the promotion of porcine epidemic diarrhea virus subunit vaccine and provided a new idea for the prevention and treatment of porcine epidemic diarrhea.The strategy of high level and long-lasting mucosal immunity induced by the subunit vaccine remains to be further studied.