Establishment And Application Of Indirect ELISA For The Detection Of Porcine Epidemic Diarrhea Virus Antibody

Porcine epidemic diarrhea(PED)caused by porcine epidemic diarrhea virus(PEDV)belongs to viral intestinal infectious diseases which could lead to typical symptoms like water like diarrhea,dehydration,vomiting in pigs.This disease mainly occurs in winter and spring,however,summer and autumn refer to sporadic cases.Pigs of all ages are susceptible,especially sucking piglets,causing huge losses to the development of China’s pig industry.In recent years,variation of PEDV epidemic strain in our country caused a decline in the effectiveness of the traditional vaccine,leading to outbreak of PED epidemic in major provinces raising pigs.Thus effective prevention and control of PEDV has become a urgent problem that needed to be solved immediately.But currently our country lacks effective pathogen detection kit,and the domestic market is still short of commercially available detection kit for PEDV antibody.Imported testing kit is expensive,not suitable for promoting the use to grassroots level.Hence it is the research intention of this test to develop a reliable and suitable detection kit for PEDV serology detection among high-volume samples.The spike protein,S protein of PEDV is an important structural protein that mediates the virus’ entry to host cells,and stimulates cells to produce neutralizing antibodies.Moreover,antibodies produced by S protein exist for a long time during the disease process,which is of great significance in the pathogen detection and epidemiological study.In this study,we use recombinant truncated S protein expressed by the prokaryotic Escherichia coli expression system as antigen,developing a ELISA method to detect antibodies of PEDV,and applying the ELISA method to detect clinical samples,which provide a reliable technical support to investigate the prevalence of PED.The content of study were as follows:1.Prokaryotic expression of truncated S protein of PEDVA pair of specific primers were designed based on the S gene sequence of PEDV CZ2014 strain published in GenBank.Parts of S gene was amplified by RT-PCR and connected to the E.coli expression system’s subcloned expression vector pET-28a.The recombinant plasmid was constructed and named 28a-S1.Then the recombinant plasmid was transformed into host strain BL21(DE3)and induced by IPTG,then identified by SDS-PAGE to recognize the recombinant protein S1,which size was about 40 KDa and expressed as inclusion bodies.After the recombinant protein was purified,western blotting analysis proved that it can be combined with PEDV positive serum,indicating that the reactogenicity of the expressed recombinant protein was good.2.Establishment of an indirect ELISA method for PEDV antibodies detection.The purified recombinant protein S1 was used to coat ELISA plates to establish an indirect ELISA method to detect antibodies of PEDV.The optimized reaction conditions were as follows:the optimal antigen coating concentration was 7.5 μg/mL,coating condition was 37℃ 2 h.The optimum blocking buffer was 5%skim milk in PBS.The optimal serum dilution was 80-fold,and the react time was 37℃ 30min.Dilution of HRP-SPA was 1:10000 and incubated at 37℃ 1h.The best react time of TMB substrate solution was 20 min.The ELISA cut-off value was 0.286,determined by measuring 33 negative serum samples.Through test the average reproducibility of this method within the batch was 3.31%,inter-assay average reproducibility was 4.32%.The method was used to detect Swine fever,porcine transmissible gastroenteritis,swine rotavirus,porcine circovirus type 2 positive sera and results were negative,indicating that this indirect ELISA test method had good reproducibility and specificity.3.Application of indirect ELISA method for detecting PEDV antibodiesImported PEDV detection kit was compared with ELISA method established by this study,and 92 PEDV serum was tested with different antibody titers simultaneously,the overall rate was 86.96%,proving that the ELISA method had good sensitivity and specificity.The established indirect ELISA method was used to detect PEDV antibodies of 564 pig serum in different ages acquired from different pig farms of Chia Tai group in Nantong,Jiangsu and Wen’s Group.Results showed PEDV total positive rate was 61.17%,at same time positive rates of pigs’ PEDV serum antibody varied from ages.Positive rate of PEDV antibody in sucking piglets’ serum was 26.47%,and positive rates of weaned pigs and feeder pigs were 36.67%,50%respectively.The positive rate of fattening pigs was 31.93%.Gestation sows,lactating sows and breeding swine had high serum antibody levels to PEDV due to vaccination.The positive rate of replacement gilts reached 85.29%.The research showed that the antibody positive rate of PEDV varied in pigs from ages and groups.Sucking piglets and fattening pigs had lower positive rates,but positive rates of pigs in other ages were higher relatively.ELISA method established by the test was used to PEDV serological tests of large sample size,providing important reference for PEDV diagnosis and antibody levels monitoring after immune,etc.