Establishment Of IgA ELISA Method And Immune Efficacy Evaluation Of Recombinant Attenuated Vaccine Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea(PED)is an epidemic disease of the highly contagious swine intestine caused by the porcine epidemic diarrhea virus(PEDV).Pigs of all ages are susceptible and exhibit different degrees of symptoms such as diarrhea and dehydration,and piglets within a week of age are more likely to experience extreme mortality from severe diarrhea,dehydration,and vomiting.Since 2010,PED has been prevalent in the country,and detection of other immune markers,causing great challenges for vaccine control and prevention in the country.The recombinant weakly virulent vaccine PEDV was constructed in our laboratory previously by replacing the S gene of the classical vaccine strain DR13 with the S gene of the domestic circulating strain using RNA site directed recombination reverse genetic manipulation.In this work,we first developed an indirect ELISA for the detection of PEDV IgA antibodies by using the recombinant S protein of PEDV as the coating antigen,and then,we evaluated the efficacy of this recombinant PEDV weakly virulent vaccine developed in the previous period of immunization of sows during the second and third trimesters by measuring the levels of IgA,Ig G,and neutralizing antibodies in serum or milk,as well as the rate of protection from passive immunization after the challenge in piglets.The main content and results of this paper are summarized as follows:1.Preparation of recombinant S protein of PEDVTo obtain PEDV s protein that could be used as the detection antigen in indirect ELISA with good reactivity,p XXGST-S plasmid transformation,inducible expression,protein purification as well as Western blot verification were performed in the paper.The results showed good immunoreactivity of the induced expressed PEDV S protein.2.Establishment of indirect ELISA for detecting PEDV IgAIn the paper,recombinant S protein was used as the coating antigen,Goat anti swine IgA HRP was used as the secondary antibody,and the reaction conditions of indirect ELISA were optimized to establish the detection method.The results showed that when the antigen coating amount was 1.28μg/100 μ50.The best results were obtained by indirect ELISA at a BSA blocking solution concentration of 1 μ g / ml,a sample dilution ratio of 1:4,and a microplate secondary antibody dilution of 1:5000.The assay has a cut-off value of 0.287,remains detectable after 32 fold dilution of positive samples,and has good specificity and reproducibility for detecting PEDV IgA antibody levels in milk after vaccine immunization.3.Evaluation of immune effect of recombinant attenuated vaccineBriefly,sows immunized in the second trimester(post gestational days 57-59)or third trimester(post gestational days 85-87)were immunized intramuscularly with recombinant PEDV attenuated vaccine(titer 107.15 TCID50 6 ml / head)and infected with the same dose of viral solution(pedv-xs12)orally to wild-type control sows for a further 15 d after initial immunization.Sera were collected from the vena cava 5 d after the primary immunization,5 d after the secondary immunization,and 10 d postpartum,and colostrum and 7 d postpartum normal milk were collected to determine IgA,Ig G,and neutralizing antibody levels in sera or milk.In addition,piglets 4 d after birth were inoculated orally with the viral solution described above(at a dose of 1597 copies / kg),body temperature and weight were measured for ten consecutive days,and Ig G antibody levels in the serum of the piglets were determined by phlebotomy and separation of serum 0 d,4 D,and 7 d after challenge.The results showed better levels of IgA,Ig G,and neutralizing antibodies in sows immunized during the second trimester compared with the other groups tested,and the survival rate of the piglets obtained was 100%.In summary,an indirect ELISA assay for PEDV IgA antibodies based on the recombinant S protein of PEDV was successfully established in this study.Combined with the IgA,Ig G,and neutralizing antibody levels and passive immune protection following piglet challenge,it was initially thought that sows vaccinated in the second trimester with recombinant PEDV attenuated vaccine would have better passive immune protection in piglets.