Antigenicity Analysis And Preparation Of Monoclonal Antiboy Of Prokaryotic Expressed N-terminus Of S Gene Of The Classical And An Epidemic Strain Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea(PED),caused by porcine epidemic diarrhea virus(PEDV),is an acute and highly contagious intestinal disease in swine.The clinical symptoms of the disease are diarrhea,vomit and dehydration.The spike(S)protein is one of the structural protein on the surface of PEDV particles.It performs a pivotal role in regulating the interactions of virus with glycoproteins receptor of specific host cell to mediate viral entry and inducing neutralising antibodies in the natural host.Phylogenetic analysis based on the nucleotide sequence of domestic and overseas PEDV S genes showed that the 5’ region of S gene of the epidemic strains have obviously changed,including deletion and insertion compared with classical strains(CV777).In this study,the immunoreactivity of N-terminus of S gene of the classical and an epidemic strain of PEDV was compared.We hope our study can provide some references for the genetic variation,differential diagnosis and immunoprotection mechanism of PEDV.The 5’ region of S gene located in 61-661bp of CV777 and 61-693bp of an epidemic strain named NB-2011 was amplified by RT-PCR and cloned into the expression plasmid pET-28a.The recombinant plasmids were named pET28a-S661 and pET28a-S673,respectively.Two fusion protein(S661 and S673)were expressed in E.coli BL21.Western-blot results indicated that S661 and S673 recombinant proteins could specificity react with porcine positive serum against PEDV but not with negative serum.The polyclonal antisera were prepared by immunization of Balb/c mice with the recombinant protein S661and S673,and the cross Dot-ELISA was conducted on the two kinds of the proteins with the antisera.The results showed that the minimum concentrations of the protein S661 and S673 with visible dots reacting with antiserum of S661 were 31.25μg/ml and 125μg/ml,respectively,and with antiserum of S673 was 62.5μg/mll equal to the two proteins.The monoclonal antibody(McAb)against S661 and S673 was prepared.The 4 positive hybirdomas against S673 named 1D7,2E11,3G9,4G5 and the 2 positive hybirdomas against S661 named 4A12,5G8 were obtained.Identification of isotypes of McAbs showed that 3G9,5G8 and 4A12 were belonging to IgGl,1D7 and 2E11 belonging to IgG2a,4G5 belonging to IgG2b.The McAbs overlying ELISA tests showed that 1D7,2E115 3G9 and 4G5 were against the same epitope of S673 and 4A12,5G8 were against the same epitope of S663.The six McAbs reacted specificly with the corresponding protein but not with the other immunogen by cross Western-blot.Indirect immunoflurescence assay showed that all six McAbs could react with Vero E6 cells infected by PEDV in which the specific immunoflurescenc were detected.The all above results shows that the antigenicity of N-terminus of S protein between the classical and an epidemic strain of PEDV are different.