Study On Subunit Vaccineofswine Epidemic Diarrhea Basedon S1 Proteincore Neutralization Epitope Region

Porcine epidemic diarrhea(PED),an acute,highly contagious intestinal infectious disease,which is caused by porcine epidemic diarrhea virus(PEDV),and mainly characterized by diarrhea,vomiting,dehydration and high mortality.Since 2010,the PEDV variant strains is widely epidemic in most provinces in China,whichcasue huge economic losses to swine industry.Vaccination is the most effective way to prevent and control PED epidemic.Currently,the PED prophylactic vaccines include multi-inactivated and attenuated vaccines,but they both have shortcomings including low protection rate,low security,high cost and so on.it is urgent to develop a more secure,economical and efficient new vaccine(especially subunit vaccine)for the control of PED.The PEDV S1 domain is the primary functional region of S surface protein that mediates PED protective immunity.Core neutralizing epitope(COE)is the most important functional region of S1 and is the smallest protective antigen for PED subunit vaccine R&D.Therefore,this study is focused on expression the COE of PEDV S1 by the Pichia pastoris expression system.To realize the antigen targeting to antigen presenting cells(APCs)and improve the immunogenicity,we further expressed the Ig G1 Fc fused COE as well.The main results were as follows:a)The COE,P1 Fc and H1 Fc fragments were amplified by PCR,then the COE-P1 Fc and COE-H1 Fc fragments were spliced by overlapping PCR.The yeast expression vectors of p PICZ?A-COE,p PICZ?A-COE-P1 Fc and p PICZ?A-COE-H1 Fc were constructed successfully;b)Western blotting result indicated that three recombinant proteins of r COE,r COE-P1 Fc and r COE-H1 Fc were expressed successfully in the Pichia pastoris expression system;c)In 42 day post immunization,indirect ELASA results showed that the three groups of experimental groups have produced a higher level of antibodies.The results of serum neutralization test showed that theneutralize antibody titers of r COE,r COE-P1 Fc and r COE-H1 Fc were 1: 160,1: 640,1: 512 respectively,and theexperimental group neutralize antibody titers of r COE-P1 Fc,and r COE-H1 Fc were higher than the r COEgroup and the PBS control group.The results showed that P1 Fc and H1 Fc have better adjuvant effects by fusion to C terminus of the COE.In this study,the fusion protein of S1 protein core neutralizing epitope was first successfully expressed by Pichia pastoris expression system.And the Ig G1 Fc fragment could be used as adjuvant to improve the immunity of PED subunit vaccine based on COE.