Prokaryotic Expression Of COE Epitope Of Porcine Epidemic Diarrhea Virus And Establishment Of Indirect ELISA Test Method

Porcine epidemic diarrhea is a highly contagious intestinal infectious disease whichcaused by porcine epidemic diarrhea virus.All ages pigs are thought to be susceptible toinfection. It occur frequently around the world and result in serious economic losses for thepig industry.Since2010,PED occur in isolation or mixed infections with other pathogens.It causesevere diarrhea of piglets of various scale pig farms.Morbidity and mortality weresignificantly higher.The vaccine is an important means of prevention and treatment ofPED.While the cultured in vitro of PEDV is difficult,virus content of vaccine tends to beinsufficient, which can cause immune failure.In addition,the PEDV attenuated vaccine forthe suckling pig exists the residual virulence. There will be the risk ofpathogenic.Therefore,development of PEDV gene engineering vaccine has importantsignificance on the prevention and treatment of PED.In addition,the PEDV diagnosticreagents used at home and abroad are mostly whole virus.But the separation and culturedin vitro of PEDV is very difficult,so a large number of antigen preparation is extremelydifficult.In summary, the test PEDV strains of the S gene popular in recent years, theprotective antigen (COE) of the conservative further genetic analysis with COEprokaryotic expression protein PEDV indirect ELISA diagnostic techniquesand to providefor the the PEDV new genetically engineered vaccine theory and material basis.This testanalysised the conservative of the protective antigen gene (COE) of S gene of PEDVstrains which is popular in recent years,established indirect ELISA diagnostic techniques ofPEDV with COE prokaryotic expression protein,and provide theoretical and material basisof the new genetic engineering preparation vaccine.Studies are as follows:(1)This test designed one pair of primers P1,P2according to PEDV CV777straincomplete gene sequence (accession number as AF353511) from GenBank,and amplifiedCOE fragment by RT-PCR.The test successfully made the target gene insert into theprokaryotic expression vector pET-32α and built recombinant expression plasmidpET-32α-COE.The recombinant plasmid pET-32α-COE was transformed into theexpression host strain E.coil Rosetta and induced to express.The SDS-PAGE result showedthat the target gene fragment was expressed in E.coil Rosetta,and the target proteinmolecular weight was about40kD.The high purity of the recombinant protein was obtainedafter extraction and purification of inclusion bodies.Western-blotting analysis showed that the recombinant protein had good immunogenicity.The study has laid material andtechnical basis for the research of diagnostic reagents and the genetic engineering vaccineof PED.(2)This test made purified recombinant protein as coating antigen,optimizedconditions of indirect ELISA and established an indirect ELISA detection method forPEDV. Detect clinical serum by this method and porcine epidemic diarrhea antibody rapiddetection kit.The results showed that this method has highly specificity,sensitivity,reproducibility and highly consistent rate.It lay the foundation for the furtherpreparation of porcine epidemic diarrhea antibody diagnostic kit.At the same time,itprovides good technical means about the diagnosis and detection of porcine epidemicdiarrhea virus.