Establishment Of An Indirect ELISA For Porcine Epidemic Diarrhea Virus Detection Based On Pichia Pastoris-expressed COE Protein

Porcine epidemic diarrhea(PED)is a severe contagious intestinal disease caused by porcine epidemic diarrhea virus(PEDV).Pigs of all ages are susceptible to infection,especially the sucking piglets(aged below 7 days),which could reach up to mortality of 90%,causing serious economic losses to pig farms.In addition,the excessive use of medicines for controlling PED on susceptible pigs leads to residue in food products and brings potential threats to food safety.Thus,it was of great importance to develop a rapid and effective detection method for PED diagnosis and vaccine motoring.Serologic tests,such as enzyme-linked immunosorbent assay(ELISA),are widely applied in the ditection of anti-PEDV antibody in sera.ELISA is becoming more important because of its high sensitivity,strong specificity,easy operating and rapid for clinical diagnosis.The CO-26 K equivalent(COE)protein(499-638 aa)regions in PEDV S protein is a highly conservative neutralizing domain and can produce PEDV neutralizing antibody.It plays an important role in immune response induced by S protein,which makes it a suitable antigen for the detection of anti-PEDV antibody.In this study,an indirect ELISA based on recombinant COE protein(r COE-i ELISA)expressed in Pichia pastoris(P.pastoris)was developed.The main results are as follows:1.Expression and preparation of r COE protein in P.pastorisThe PEDV COE gene was inserted into P.pastoris expression vector p PIC9 K,and the recombinant plasmid p PIC9K-COE was acquired.After the p PIC9K-COE linearized by Sac I and transformed into P.pastoris GS115 by electroporation,the positive recombinant colony was screened for methanol-induced expression,yielding 3.3 mg/L of r COE protein.SDS-PAGE showed that the r COE protein with an approximate molecular weight of 22 k Da was obtained.The purified r COE protein was well recognized by anti-PEDV positive serum when using it as the primary antibody in western blotting,indicating that the well reactogenicity of r COE protein.2.Development of r COE-i ELISATo establish an indirect ELISA method based on r COE protein,different reaction conditions were optimized.The optimal antigen concentration was 1 μg/m L,coating for 12 h at 4℃.It was found that 5% skimmed milk was the optimal blocking buffer and the best blocking time was 1 h at 37℃.The optimal serum dilution multiple was 1:400,and the reacting time was 45 min at 37 ℃.The optimal condition of secondary antibody was10000-fold diluted for 30 min incubation at 37℃,and the optimal stopping time was determined as 10 min.Under the optimized conditions,the cut-off value of r COE-i ELISA was calculated as sample to positive(S/P)ratio of 0.12 through ROC analysis using serum neutralization test as standard.The r COE-i ELISA was demonstrated a relative sensitivity of94.4% and specificity of 92.6%.3.Characterization of r COE-i ELISAThe established r COE-i ELISA showed no cross reactivity to three other swine viruses including porcine reproductive and respiratory syndrome virus(PRRS),classical swine fever virus(CSFV)and pseudorabies virus(PRV).The sensitivity test showed that PEDV-positive serum could still be accurately diagnosed as positive at 1600 times dilution.Meanwhile,the variable coefficients(CV)of intra-assay ranged from 1.58% to 5.92%,whereas the inter-assay CV was between 0.86% and 6.85%.All results were clearly below the in-study validation acceptance criteria of 15%.Moreover,244 clinical serum samples were tesetd with an accuracy of 97.54%.Comparing the test results with two commercial kits,it was found that the overall agreement of the r COE-i ELISA with two commercial kits were 95.08% and 92.62%,respectively.In conclusion,the soluble r COE protein expressed by P.pastoris showed a well antigenic activity and the developed r COE-i ELISA exhibited great sensitivity,specificity and repeatability as well as high accuracy in application for vaccinated sera samples.It suggested the r COE-i ELISA may be a reliable method for detecting antibody to PEDV in the sera of infected or vaccinated pigs.The study laid a foundation for further preparation of a diagnostic kit for antibody detection of PEDV.