Preparation Of Polycional Antibody Of The Truncated S1 Protein Of Porcine Epidemic Diarrhea Virus And The Establishment Of Indirect ELISA

Porcine epidemic diarrhea（PED）is an acute,destructive,highly contagious intestinal disease caused by porcine epidemic diarrhea virus（PEDV）that characterized by severe watery diarrhea,vomiting,dehydration,low or no food and higher mortality in suckling piglets,which results in significant economic losses in the swine industry worldwide.The S glycoprotein of PEDV was recognized as a class I transmembrane glycoprotein,which could be divided into the S1 and S2 regions that responds for viral receptor binding and host-virus fusion,respectively.The S1 regions play a major role in the specific,high-affinity binding of cell receptors and induction of the neutralizing antibody.Therefore,the PEDV S1 domain is known to be an ideals candidate for the development effective vaccine and diagnostic assays.In this study,the target fragment of truncated PEDV S1 was obtained by PCR amplification using the c DNA of the SX-NQ PEDV mutant strain as a template,and was cloned into prokaryotic expression vector p ET-32a,then transferred into E.coli BL21（DE3）to express,and identified by SDS-PAGE and western blot.The optimized expression conditions were:25℃,with 1mmol/L IPTG shaking for 4h.The target protein existed in inclusion body.The protein was purified by Ni-NTA and refolded by gradient dialysis to obtain high purity and bioactive protein.The polyclonal antibody was obtained by immunized mice with the obtained protein.The antibody titer of S1 protein in the serum of mice was 1:200000 after three times immunization.The antibody was identified by IFA and western blot.The results showed that the polyclonal antibody of truncated S1 protein could react specifically with PEDV S protein expressed in baculovirus.Using the purified recombinant protein as detection antigen,the indirect ELISA detection method of PEDV antibody was established.According to the experiment,the optimal detection conditions for ELISA were determined as follows:the optimal coating antigen was4μg/m L,4℃overnight.Block the plate with 2.5%dry milk PBS’T at 25℃for 90 minutes,the serum was diluted in the ratio of 1:100 at 25℃for 90 minutes,and the HRP Goat@Swine Ig G was diluted by 1:2500.The OD_450nmvalue was read after 90 minutes.The critical value of this method is determined to be 0.177.The method has the advantages of high specificity,good stability and good coincidence rate compared with commercial kit.This method was used to detect 384 pig serum antibodies,the positive rate of the samples was 83.3%.The applicability of the method is preliminarily proved.In conclusion,the PEDV truncated S1 protein with high purity was obtained by prokaryotic expression,the polyclonal antibody was obtained by immunized mice,and the indirect ELISA diagnostic method was established.It provides a basis for the development of clinical diagnostic kit for PEDV.