The Molecular Epidemiological Survey Of Porcine Epidemic Diarrhea Virus And Construction Of Vero-APN Cell Line

Porcine epidemic diarrhea(PEDV)is one of the main causes of dirrhea in pigs.It has caused severe economic losses to world as well as China since 2010.PEDV belongs to RNA virus and has a high variation,this is why the traditional vaccine cannot provide good protection for piglets.PEDV was difficult to be isolated so the study of its etiology is very slowly.PEDV is sensitive to Vero cells only but Vero cells don’t belong to porcine cells.pAPN is the receptor of PEDV but Vero cells lack of this receptor.In this study,PEDV was detected from the materials that sent to the laboratory from 2014-2015,and 7 PEDV positive samples were selected to amplify the S gene and sequenced to have a better understanding the variation of prevalent strains.pAPN was expreesed in prokaryotic expression system and prepared the anti-pAPN rabbit polyclonal antibody.A Vero cell line expressing pAPN was successfully constructed,which laid the foundation for the isolation of PEDV and the study of pAPN.The details are as follows:1.PEDV detection and genetic variation analysis of PEDV S geneA total of 104 samples from small intestinal tissue of diarrheal piglets were detected by RT-PCR from 2014 to 2015.The results showed that 42 samples were positive for PEDV.The sequence of S gene from 7 PEDV positive samples were cloned and analyzed by comparing with the referenced PEDV strains in GenBank.The results suggested that the nucleotide and amino acid homologies of the seven S genes were 98.1%to 99.2%and 97.6%to 99.1%,respectively.The seven PEDV S gene sequences shared 94.0%to 94.2%and 93.9%to 94.1%nucleotide identities and the amino acid sequences similarity were 93.1%to 93.6%,92.5%-93.1%respectively comparing with the reference strains CV777 and DR13.The phylogenetic tree based upon sequences of S gene suggest the PEDV strains could be assigned to three genetypes and the seven PEDV strains all belong to type III,which revealed a close relationship with the PEDV isolated in China from 2012 and different from traditional foreign strains(the CV777 and DR13 attenuated vaccine strain)and early Chinese strains.2.The expression of pAPN in prokaryotic expression system and the preparation of the polyclonal antibodies against pAPNAPN was the cellular receptor of variety of coronavirus as well as PEDV.APN of porcine(pAPN)was amplified by PCR in this study and cloned into the prokaryotic expression vector pET-32a.The obtained the recombinant plasmid pET-32a-pAPN was transformed into E.coli BL21 strain.The recombinant protein was identified by SDS and Western-blot.Result showed the recombinant protein was expressed and existed in the form of inclusion body.The recombinant protein was purified by inclusion body lotion and was used to immunize rabbits after emulsified with Freund’s adjuvant.The rabbit was slaughtered after the third immunization.The serum was separated and its antibody titer was measured by ELISA method.The rusults showed the protein had a better antigenicity and the serum contained high titer of specific antibody.The study laid the foundation for the research of pAPN in the later stage.3.Construction of a cell line expressing pAPN and the activity of pAPNIn order to study the effect of pAPN on the replication of PEDV and enhance PEDV infection in cells.In this study,we cloned the complete pAPN sequence into the lentiviral vector pLVX-mCherry by homologous recombination.The recombinant plasmid pLVX-mCherry-pAPN transfected 293T with lentiviral packaging plasmids PLP1,plp2,PLP/VSVG.After packaged lentivirus containing the target gene in 293T cell the recombinant virus was harvested and infected to Vero cells.The cells expressing pAPN were selected by cultured in nutrient solution containing puromycin.The expression of APN in the cell line was identified by RT-PCR and IFA.A monoclonal cell line was successfully obtained using single cell cloning technology and was named Vero-APN-B6.The susceptibility to PEDV of this cell line comparing with the normal Vero cells was detected by the methods of IFA and determination of TCID50.The results showed that the gene of APN was integrated with the genome of the Vero cell and the cell line was more susceptible to PEDV than the normal Vero cells.This cell line is expected to be applied to improve the proliferation of PEDV in cells and the isolation of PEDV.In this study,we investigated the variation of PEDV S gene to understand the difference of antigenic sites between variant strains and traditional strains,which laid the foundation for the study of pathogenic mechanism and prevention of PEDV.pAPN polyclonal antibody was prepared to identify the cell line expressing pAPN and this will provide conditions for the study of pathogenic characteristics and pAPN activities.