The Preparation Of Bivalent Inactivated Vaccine Against PDCoV-PEDV And Immunogenicity Of The Vaccine In Mice

Porcine epidemic diarrhea?PED?caused by porcine epidemic diarrhea virus,is an acute contact intestinal infection characterized by vomiting,watery diarrhea and dehydration.PED is one of the major viral infectious diseases that seriously restrict the development of the pig industry.Since 2010,PEDV has appeared on a large scale in pigs across China,vaccine prepared with CV777 strain as antigen is not effective after immunized,the vaccinated farm still had this disease.In addition to feeding management factors,variation of endemic strains may be an important cause of immune failure.In 2012,the Porcine deltacoronavirus?PDCoV?was first discovered by Hong Kong scholars in China,then subsequent reports had also appeared in other provinces.PDCoV and PEDV are coronaviruses with very similar clinical symptoms,and there are no commercial vaccines for PDCoV.This study is based on PEDV and PDCoV in Hebei,prepared PDCoV and PEDV inactivated bivalent vaccine,inoculate Balb/c mice to study vaccine immunogenicity.The specific research content is as follows:In order to increase the antigen content of the inactivated vaccine,this study optimized the in vitro culture conditions of PEDV and PDCoV.Respectively from optimal virus inoculation titer,maintenance fluid dosage after virus adsorption,best time to harvested virus.The experimental results showed that when the PEDV HB-HS strain was in the titer of 10^4.35 TCID₅₀/mL,added 8 mL of maintenance solution,harvested virus after60h,virus titer reached to 10^5.91 TCID₅₀/mL.When PDCoV HB-BD strain was in the titer of 10^4.52 TCID₅₀/mL,added 8 mL of maintenance solution,harvested virus after 72h,virus titer reached to 10^5.57 TCID₅₀/mL.In vaccine preparation,inactivated the two viruses using final concentration of 0.3%formaldehyde,prepared of a water-in-oil?W/O?inactivated vaccine by white oil Span,which was an immunoadjuvant.The residual virulence,physical properties,sterility and safety test results after inactivation of the inactivated vaccine are in line with the standards in the Chinese Veterinary Pharmacopoeia.In order to evaluate the vaccine immune effect,used pET-32a-PDCoV S1 positive expression plasmid and pET-32a-PEDV S1 positive expression plasmid,prepared of PEDV recombinant S1 protein and PDCoV recombinant S1 protein by prokaryotic expression system.We made two recombinant proteins as antigens,determined the optimal concentration of antigen by square matrix titration,and established two indirect ELISA method for detecting PEDV S1 protein IgG antibody and PDCoV S1 protein IgG antibody in mouse serum after optimized the reaction conditions.Detected mouse negative serum by two ELISA methods,calculated the respective negative-positive thresholds by the formula.The ELISA antibody titer of the serum to be tested was determined by detecting the serum maximum dilution ratio when the OD_450nm of the serum sample is greater than or equal to the negative value of the negative.In term of vaccine immunogenicity test,inoculated Balb/c mice with different immunization doses of inactivated vaccine,collected of blood in stages before the first exemption and after the first exemption,determinated of specific antibody titers in serum,drawn antibody growth curve.The optimal immune dose of the vaccine was determined as0.2mL by the antibody growth and decline curve.In the immunogenicity test results of different inactivated vaccines at the optimal immune doses,inactivated bivalent vaccine group PEDV ELISA antibody titer,maximize?1:1006?after 4 weeks of the first time immune,higher than PEDV commercial vaccine group?1:654?,lower than PEDV inactivated vaccine group?1:1252?.Antibody titer began to decline after 5 weeks of the first time immune,but the antibody titer of inactivated bivalent vaccine group was still higher than that of the commercial vaccine group.PDCoV ELISA antibody titer reached peak?1:982?after 4 weeks of the first time immune,but lower than PDCoV inactivated vaccine group?1:1150?.Antibody titer was still high after 6w of the first time immune?1:870?,then the antibody titer began to decline rapidly.The antibody growth curve indicated that the level of antibody produced by the inactivated bivalent vaccine group is lower than that of the inactivated vaccine group,this was due to differences of the effective antigen content of different vaccines at the same immunization doses.Experimental data showed that the preparation of PDCoV-PEDV inactivated bivalent vaccine could stimulate high-level specific antibodies in mice.