Examination On S Protein Fusion Epitope Nucleic Acid Vaccine Of PEDV And TGEV

Both porcine epidemic diarrhea virus(PEDV)and transmissible gastroenteritis virus(TGEV)can infect pigs of all ages,which has a serious impact on the wordwide pork industry.Particularly,the mortality rate of suckling piglets is extremely high.Spike protein of PEDV and TGEV,which is the main target for inducing the host to produce antiviral neutralizing antibodies,is a glycoprotein located on the surface of virus.Currently,the S gene is the most important targeting antigen gene for PEDV and TGEV vaccines.However,because of the S genes of PEDV and TGEV is susceptible to variability,resulting in reduced immune protection efficacy of current inactivated and attenuated vaccines,development new safer and higher efficient vaccines is still urgent.Both the PEDV COE gene and TGEV A and D antigenic sites in the S protein is a highly conservative neutralizing domain and has better immunogenicity.Nucleic acid vaccines have safer and chronergy,compared with subunit and attenuated vaccines,which can better stimulate the humoral and cellular immune responses in the host.The nucleic acid vaccines of PEDV and TGEV not only stimulate specific neutralizing antibodies but also induce cellular immune responses in mice.In order to develop nucleic acid vaccine,enhancement the immunogenicity of antigens is the crux of the matter.Escherichia coli heat-labile enterotoxin B subunit(LTB),which enhances the immune response of the host as a novel vaccine adjuvant.So far,no relevant studies have been reported to use LTB as an adjuvant for TGEV and PEDV nucleic acid vaccines.In our study,a nucleic acid vaccine carried of the S gene of TGEV and PEDV fused with the LTB gene as adjuvant was developed.The research methods and results are as follows:In this study,the COE gene(AF353511.1)of PEDV(CV777)and the A and D antigenic site genes of TGEV S gene(AJ271965.2)were fused by a flexible Linker sequence as an antigen gene,and the gene of Escherichia coli heat-labile enterotoxin B subunit(S60731.1)was used as immune adjuvant.The eukaryotic plasmid of pLTB carrying LTB gene,pPT carrying PEDV COE with TGEV S protein A?D site fusion genes,and pPT-LTB recombination carrying PEDV COE with TGEV S protein A?D fusion site genes and LTB gene using the bicistronic eukaryotic expression vector pIRES as a plasmid expression vector was constructed.To investigate the expression of the positive plasmid in vitro,plasmids was transfected into BHK-21 cells and identified by Western Blot.The results of this experiment showed that the recombinant protein can be successfully expressed in vitro.To research the immune effect of recombinant plasmid,we studied by intramuscular injection,using Kunming mice as an animal model.Six to eight-week-old female Kunming mice were randomly allocated into 6 groups,20 in each group,respectively PBS group,pIRES(blank vector)group,pLTB group,pPT group and pPT-LTB group,and PEDV/TGEV live vaccine(YM)group,and intramuscularly with 100 ?l of recombinant plasmid per dose for three times at two-week intervals.For the detection of fluid and cellular immunity levels,peripheral blood and spleen were collected at each time of each group.The YM group has the highest value,followed by the pPT-LTB group,in the T lymphocyte proliferation assay,specific cytotoxic T lymphocyte assay,specific IgG and neutralizing antibody levels.Among with the PBS,pIRES,and pLTB groups,there was significant difference between pPT and pPT-LTB.When compared with pPT and pPT-LTB,pPT-LTB were significantly higher(P<0.01).In the IFN-? and IL-4 assays,pLTB,pPT and pPT-LTB were extremely significant difference(P<0.01)compared with the PBS and pIRES groups.In the IFN-? content test,compared with the pLTB group,there was a significantly higher difference between the pPT and pPT-LTB groups(P<0.01).In the IL-4 content test there was a significant difference in pPT and pPT-LTB(P < 0.05).In our study,the IFN-? content test in the pPT and pPT-LTB groups were significant difference,and there was no difference in their levels in the IL-4 content test.The results indicated that LTB can significantly enhance the immune effect of PT eukaryotic expression plasmid as an adjuvant molecule.To the best of our knowledge,this is the first study used the eukaryotic expression vector pIRES to express the fused PEDV COE gene together with the TGEV S A and D gene with the LTB adjuvant gene.The constructed recombinant nucleic acid vaccine has obvious immunogenicity.The results showed that in this study,LTB can be used as an adjuvant molecule to significantly improve the antiviral ability of the organism,thereby improving the cellular and humoral immune responses.All of these findings provide theoretical basis for the research of the novel PEDV/TGEV binary nucleic acid vaccine.