Construction And Expression Of Spike Protein Of PEDV In Insect Cells And Establishment Of An Indirect ELISA For Detecting Antibodies To PEDV

Porcine epidemic diarrhea(PED) is a severe infectious disease which is caused by Porcine epidemic diarrhea virus(PEDV) with diarrhea,vomit and dehydration.This disease mainly occurs in winter from December to February next year,and may also occur in summer.It can infect the enteric and respiratory tissues of newborn piglets and cause fatal diarrhoea in young pigs resulting in mortality of nearly 100%.PED has become one of the most important virulent diarrhoeal diseaseses in pigs.In recent years,the popular region had expanded gradually.Therefore,it is necessary to establish an effective and fast method to detect PED.But PED has no obvious difference from pig infectious gastroenteritis(TGE) in clinical symptoms,epidemiology,pathological change and so on.At present,the main diagnosis methods of PED are the immuno-electron microscope,the immunity fluorescence method,the indirect blood congeal,the RT-PCR and the ELISA.The ELISA operation is more simple than other detecting methods which need the specific bench-scale equipment to be able to complete,so the applications of others have some limitations.This research expects to establish a sensitive ELISA to detect the antibodies of PEDV through the gene clone and the bac-to-bac system.The PEDV S protein is the major inducer of PEDV-neutralizing antibodies and it mediates binding of PEDV to its cellular receptor,therefore it is better to choose the S gene to be the goal gene.According to GenBank,a pair of primers was designed.The DNA fragment was amplified by RT-PCR and was cloned into the contribution vector pFastBac1 to get the recombinant plasmid pFastBac1-PS1.Plasmid pFastBac1-PS1 was then introduced into E.coli DH10Bac,which included a shuttle vector,Bacmid.By site-specific transposition,S gene was integrated into bacmid,and a recombinant shuttle vector was constructed,named Bacmid-PS1.The recombinant bacmid,which was identified by PCR, was transfected into Sf9 insect cells with Transfast Transfection Reagent Kit.The cell CPE could be observed within 5 days after transfection.And the pure recombinant baculovirus was obtained by plaque.Expression of PS1 protein(about 47KD) was examined and identified by RT-PCR,IFA,SDS-PAGE and Western-blotting.The recombinant PEDV S protein expressed in the Baculovirus Expressing System was used as the antigen for establishing an indirect enzyme-linked immunosorbent assays(ELISA).The sample is positive when P/N≥2.2.