Establishment And Preliminary Application Of Indirect ELISA For Detection Of Porcine Epidemic Diarrhea Virus IgG And IgA Antibodies

Porcine Epidemic Diarrhea(PED)is an acute,highly-contact enteric infectious disease caused by vomiting,watery diarrhea and dehydration caused by Porcine epidemic diarrhea virus(PEDV).Since the fall of 2010,PEDV has been widely spread throughout the country,which has brought huge economic losses to China's pig industry.For the prevention of PED V,vaccines are mainly used mainly for vaccines.However,due to different vaccination methods in various farms.Because of the difference in the quality of the vaccine,although the PED vaccine is inoculated,the antibody level after vaccination is often not enough,so the detection of antibody levels after PED vaccine immunization is particularly important.In this experiment,purified PEDV whole virus antigen and recombinant N protein were used as coating antigens,and an indirect ELISA method was established to detect PEDV IgA antibody in sow colostrum and PEDV IgG antibody in pig serum.The contents of this study include the following section:1)Preparation of PEDV purified whole virus antigen and recombinant N protein antigenFirstly,the recombinant plasmid pET-28B-N was transformed into E.coli Rosetta(DE3)competent cells and induced by IPTG.SDS-PAGE electrophoresis analysis confirmed that the recombinant protein was soluble.The induction conditions were When the concentration of 0.5 mmol/L IPTG was added at 15? for 24 h,the protein expression was the highest.The concentration of recombinant N protein was 1.23 mg/mL by BCA method.The expression product was purified by His-tag nickel column and then analyzed by Western-blot.The results indicate that the expressed recombinant protein is capable of specifically reacting with PEDV-standard positive serum.Next,PEDV cell culture medium was collected,treated with 10%volume of Triton X-100,and purified by ultracentrifugation to obtain PEDV whole virus antigen,and the concentration was 3.95 mg/mL by BCA method.2)Establishment of an indirect ELISA method for detecting PEDV IgA antibodies in sow colostrumAn indirect ELISA method for detecting PEDV IgA antibodies in sow colostrum was established by purifying PEDV whole virus antigen and recombinant N protein as coating antigens respectively.The optimal reaction conditions were optimized for the PEDV whole virus.The antigen and recombinant N protein were both 2 ?g/mL,the optimal blocking solution was 5%skim milk powder blocked at 37 ? for 2 h,the optimal dilution ratio of the milk samples was 1:30 and 1:10,respectively,and the reaction was carried out at 37 ?for 60 min;IgA-HRP working concentration was 1:10000,reaction at 37 ? for 60 min;TMB reaction at 37 ? for 15 min.Two methods were used to detect porcine reproductive and respiratory syndrome virus,porcine transmissible gastroenteritis virus,swine fever virus,porcine pseudorabies virus and foot-and-mouth disease virus positive samples.The repeat coefficient of variation of both methods was less than 10%.Compared with BIONOTE commercialized kits,the coincidence rates were 92.31%and 88.46%,respectively,which proved that both methods have good specificity,repeatability and high coincidence rate,and can be used for PEDV antibody detection and epidemiological investigation.3)Establishment of an indirect ELISA method for detecting PEDV IgG antibodies in serumPurified PEDV whole virus antigen and recombinant N protein were used as coating antigens to establish an indirect ELISA method for detecting PEDV IgG antibody in pig serum.The optimal reaction conditions were as follows:PEDV whole virus antigen was 1 ?g/mL,recombinant N protein was 4.92 ?g/mL,the optimal dilution ratio of serum samples was 1:100 and 1:50,and the reaction was carried out at 37? for 60 min;the concentration of goat anti-porcine IgG-HRP was 1:5000,and the reaction time was respectively Reaction at 37 ? for 60 min and 37 ? for 30 min;TMB at 37 ? for 15 min;two methods for detection of porcine reproductive and respiratory syndrome virus,porcine transmissible gastroenteritis virus,swine fever virus,pseudorabies virus and foot-and-mouth disease virus positive samples Both were negative.The reproducibility coefficients of the two methods were less than 10%.The minimum detection limits were 1:1600 and 1:800,respectively.Both methods can provide a basis for PEDV diagnosis and epidemiological investigation.In summary,this study used porcine epidemic diarrhea virus to purify whole virus antigen and recombinant N protein as coating antigen,and successfully established an indirect ELISA antibody detection method,which provided PEDV serological diagnosis,immune antibody detection and immunological investigation.Simple and fast means of detection.It also lays the foundation for the development of subsequent kits.