The Expression Of Tandem Epitopes Of S1 Gene Of Porcine Epidemic Diarrhea Virus And Evaluation Of Immunization Efficacy

Porcine epidemic diarrhea(PED)is an acute and highly contagious enteric disease of swine caused by porcine epidemic diarrhea virus(PEDV).PED is characterized by severe watery diarrhea,vomiting,dehydration,and high mortality in suckling piglets.In this study,the complete sequence of the S gene of PEDV-HN13 strain was determined.The multiple epitopes in the S1 gene were ligated in tandem for prokaryotic expression and eukaryotic expression.Finally the two kinds of expressed proteins were evaluated for humoral immunity.The specific research contents are as follows:1.The sequence analysis of S gene of PEDV-HN13 strainThree pairs of overlapping primers were designed to amplify PEDV-HN13 S gene according to the full-length genome sequence of S gene of the PEDV CV777 strain.Using RT-PCR amplification technique,target gene was amplified,and then the fragment was cloned into pGEM-T easy vector;transformed into DH5a competent cells.The positive clones were sequenced,and the sequence of each fragment was determined,and the entire sequence of the S gene was spliced.This strain was compared with other 24 PEDV strains reported at home and abroad.The phylogenetic tree was drawn using MEGA 5.0 software.The results showed that 25 PEDVs can be divided into two gene groups,namely G ? group and G ? group:G ? group is mainly consisted of classical strains,G ? group is mainly consisted of mutant strains in recent years,and PEDV-HN13 belongs to G ? group.2.Prokaryotic expression of partial S1 antigenic site tandem genes in PEDVThe S1 protein of PEDV-HN13 strain was analyzed by bioinformatics,and two regions with strong antigenic epitopes were selected.Two pairs of primers were designed for these two regions,and the tandem gene was amplified by overlap extension PCR,named PEDV-S1-F;The F gene was cloned into the pGEM-T Easy vector and sequenced to show agreement with the original sequence.The digested target fragment was cloned into the prokaryotic expression vector pGEX-6P-1 to obtain the recombinant expression plasmid pGST-PEDV-S1-F,and induced to express in BL21 competent cells.After induction by IPTG,the recombinant protein was successfully expressed,and the size of the protein was 36KD.After identification by SDS-PAGE and Western-blot,the target protein was present in the supernatant.Purification using a GST purification kit yields a single,high purity recombinant protein.3.Eukaryotic expression of partial SI antigenic site tandem genes in PEDVAfter sequencing,the positive plasmid eukaryotic expression pFast-Bac-HTA containing the tandem gene was double-digested and ligated after gel recovery.The ligation product was transformed into DH5a competent cells and the recombinant plasmid pFast-PEDV-S1-F was obtained;and it was transposed in DHlOBac competent cells,and screened and purified by blue-white spot to obtain recombinant shuttle vector plasmid Bacmid-PEDV-S1-F.The correct positive plasmid was identified by PCR was transfected into Sf9 insect cells,and the virus was harvested at 27 ? for 7 days.After three times of virus proliferation,a high titer of recombinant baculovirus was obtained.The recombinant rBac-PEDV-Sl-F baculovirus was analyzed by IFA,SDS-PAGE and Western-blot.The results showed that the recombinant rBac-PEDV-S1-F baculovirus was expressed in Sf9 insect cells.4.Evaluation of immune effects of proteins expressed by two different systemsRecombinant S1 protein immunogen and S1-displaying baculovirus particles immunogen were prepared and then subcutaneously immunized Balb/c mice.Mice were immunized three times with interval of 14 days.At 14,28 and 42 dys post primary Immunization,serum samples were collected from mice.Humoral immunity produced by immunogen-stimulated mice was examined by indirect immunofluorescence(IFA),micro cell culture neutralization experiments.The results showed that the specific antibody against PEDV was induced in the serum,the antibody titer reached 1:200,and it had a certain neutralizing ability,and the neutralizing titer reached 1:8.The rBac-PEDV-S1-F recombinant baculovirus induced serum has an IFA titer of 1:800 and has a certain neutralizing capacity,and the neutralizing titer is also 1:8.In this study,the antigenic sites of PEDV-HN13 S1 protein were expressed in tandem in prokaryotic and eukaryotic expression systems for the development of PEDV vaccine,and some preliminary results were obtained,which laid the foundation for the development of PEDV genetic engineering vaccine.