Establishment Of Indirect ELISA And Preliminary Application For Serum Antibody Against Porcine Epidemic Diarrhea Virus Based On Recombinant N Protein

Porcine epidemic diarrhea(PED)is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV),The main symptoms of swine infection were vomiting,fever,dehydration and severe diarrhea.The incidence of porcine epidemic diarrhea is mainly concentrated in winter,which can infect pigs at different stages,but suckling piglets are the highest,up to 100%,which cause huge economic losses to the pig industry around the world.At present,there is no effective prevention and control measures,studies have shown that vaccinated pigs still have outbreaks of PED,by 2010 this phenomenon is more serious.Therefore,it is necessary to establish a diagnostic method for the detection of PEDV antibody levels.In this study,the PEDV N gene was cloned and expressed in prokaryotic expression system.The purified recombinant N protein was used as antigen to establish the indirect ELISA for detection of serum antibody level of PEDV,which was preliminarily applied in clinic and the main results of this study are as follows:1.A total of 5 small intestinal specimens of pigs suspected to have died of PEDV infection were collected in some areas of Shaanxi province.The N,S and M genes of 5 samples were amplified by RT-PCR were named 1-SL,2-BJ,3-YL,4-WN and 5-HZ respectively.Homology analysis,the 5 strains of S,M and N genes with the strain CV777,S gene was lowest,the nucleotide similarity was 94.9%~99.2%,amino acid sequence similarity was 94.9%~99.7%.Followed by the N gene,the similarity was 95.1%~99.9% and the amino acid sequence similarity was 96.2%~100.0%.The homology of M gene was highest,and the similarity of amino acid sequence was 96.2%~100%.Genetic phylogenetic tree analysis showed that the S,M and N genes of the 5 strains were relatively far from the CV777 of the vaccine strain.The genetic distances of the 5 strains were different.2.A pair of specific primers for N gene were designed,extracted the virus RNA,The reverse transcription cDNA of virus RNA was used as template,and the target gene was amplified by PCR and recovered.The N gene was cloned and expressed in prokaryotic form,and the recombinant plasmid pET-28a-N was transformed into Escherichiacoli BL21.The recombinant protein was about 58 kD,and was soluble protein by SDS-PAGE.The lysis supernatant was purified by high quality nickel column,and the protein with large amount and high purity was obtained.The pure protein had good specificity detected with PEDV positive sera by Western blot.The pure N protein was used as coated antigen,and the optimal conditions of the ELISA were: the amount of antigen was 0.5 ?g/mL per hole,the sera were diluted as 1:200,1 h,the enzyme-labeled antibody was diluted as 1:5000,1 h,the developing time of TMB was 25 min.The sensitivity,specificity and reproducibility of the ELISA method were satisfactory.The clinical detection of 164 serum samples showed that the effect was good.