Expression Of Fragmental Spike Gene Of Porcine Epidemic Diarrhea Virus,Antibody Preparation And Preliminary Establishment Of An Indirect ELISA

Porcine epidemic diarrhea(PED)is an acute,highly contagious and devastating viral enteric disease and it has a high mortality rate in suckling pigs.The causal agent is porcine epidemic diarrhea virus(PEDV)belonging to the Order Nidovirales,the family Coronaviridae and the genus Alphacoronavirus.Its genome is a 28 kb single-stranded positive sense RNA.PED has outbroken worldwide,but there is still no effective method to diagnosis and control the severe disease.The aim of the research was to establish an ELISA for detection of positive sera of PEDV by expression of PEDV protein and prepare polyclonal or monoclonal-antibodies for detection of the pathogen.Studies are as follow:1 Synthesis of fragment S gene of PEDV and obtaintion of the recombinant plasmidsBased on S gene analysis of PEDV wild strains,the gene segment coding502-641 amino acid of S1 protein were selected for expression in E.coli.The expressed gene cordons were optimized based on Escherichia coli bias and the 417 bp gene was synthesized by overlap PCR.The gene fragment was cloned into two prokaryotic expression vectors to obtain the recombinant plasmids pET32a-S417?PXXGST-S417.2 Expression and purification of the recombinant plasmidsWe translated the recombinant plasmids pET32a-S417?PXXGST-S417 into BL21 for expression of two fused proteins with His(His-S417,0.1 mg/mL)and GST(GST-S417,0.3 mg/mL)tags respectively,and the purity of the two fused proteins is90% and 95%.3 Preparation of polyclonal antibodyPurified GST-S417 was mixed with adjuvant as antigen to immune rabbit for obtaining polyclonal antibody.Purified His-S417 was employed as detective antigen to monitor the titers of the antibody,which reached about 1:10000 after the third immunization.The results confirmed good immunogenicity of the expressed S protein segment.4 Establishment of an indirect ELISAThe purified GST-S417 was used as coating antigen to establishe an indirect ELISA.The following parameters were decided for the indirct-ELISA: the optimal coating concentration was 0.32?g/well,the serum dilution was 1:800,and the HRP-IgG dilution fold was 1:3000.The criterion was 0.340.This ELISA assay isreproducible and specific.The rate of the coincidence is 72.7%.This study laid a theoretical foundation for research on PEDV diagnostic kit.