Construction and Identification of a Triple Vaccine of PEDV,TGEV and PDCoV S Protein Epitope Gene
[Objective]The aim of this experiment was to construct a triple epitope vaccine against Porcine epidemic diarrhoea virus(PEDV),Porcine transmissible gastroenteritis virus(TGEV)and Porcine delta coronavirus(PDCoV)simultaneously to prevent swine-associated diseases caused by the above pathogens.[Method]In this study,immunoinformatics online softwares were used to predict and analyze the B and T cell epitopes of the S protein of three types of Swine enteric coronavirus(SECoVs).A new epitope peptide named PPT was constructed.PPT was subjected to bioinformatics analysis using online softwares such as ExPASy,VaxiJen,TMHMM,SOPMA,SOLpro and AlphaFold2.The immune response was simulated using the C-IMMSIM online software.PPT was connected to the COE sequence of PEDV,the SAD sequence of TGEV,and the CTD sequence of PDCoV through T2A and cloned into the eukaryotic expression vector pEGFP-N1.After PCR and double enzyme digestion identification,the obtained recombinant plasmid was transfected into HEK293A cells.DAPI staining,CCK8,RT-PCR and Western blotting tests were performed to verify the expression of the recombinant plasmid in vitro.[Result]The constructed epitope protein PPT consisted of 17 epitope peptides.According to the bioinformatics analysis of software,the protein was a non-transmembrane protein with stable structure,antigenicity,solubility,high hydrophilicity,and no allergenicity.The results of C-IMMSIM showed that PPT could increase the number of DC cells in the organism,induce B and T cells immune responses,and stimulate the levels of immunoglobulins IgG,IgM and cytokines interferon-γ(IFN-γ)and interleukin-2(IL-2).PCR and double enzyme digestion identification of the recombinant plasmid showed specific target bands at 3 359,2 375,1 064,944,764 and 467 bp,corresponding to the expected sizes.After the transfection of the recombinant plasmid into HEK293A cells,green fluorescence was observed.RT-PCR amplification yielded bands matching the size of the target gene at 3 359,2 375,1 064,944,764 and 467 bp,respectively.CCK-8 assay results indicated that the recombinant plasmid had no significant cytotoxic effect on cells.Western blotting analysis showed specific bands at 31.7,16.1,37.9 and 27.5 ku,corresponding to the molecular weights of the target genes,confirming the successful expression of the recombinant plasmid in HEK293A cells.[Conclusion]Based on the PPT epitope protein designed by computer software analysis,the triple vaccine was successfully constructed and expressed in vitro,which provided a basis for evaluating the immunological effect of the triple epitope vaccine PEDV-TGEV-PDCoV.
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