| Porcine deltacoronavirus(PDCoV)is a newly discovered coronavirus causing newborn piglets acute watery diarrhea,vomit,dehydration and death.Infection of this virus has emerged in the global pig areas,raising swine disease and potential public safety problems have gradually attracted attention.In this study,The S gene of PDCoV Sichuan strain was cloned and the bioinformatics analysis was carried out.Subsequently,the S1 region protein was expressed and then used for the research of construction of monoclonal antibody preparation.These studies provide material for the subsequent study of PDCoV diagnosis and prevention1.Clone and bioinformatics analysis of PDCoV S geneThe S gene fragment of PDCoV from a fecal sample from a pig farm in Sichuan province was amplified,cloned into T-vector by RT-PCR method by two pairs of primers according to the S gene sequences in NCBI.The gene sequencing and mutation analysis was subsequently performed.The result showed that the S gene was 3480 bp in length,it has 98.74%gene similarity comparing with S gene of CH/Sichuan/S27/2012 strain.The amplified PDCoV S gene was named as CHN-SC2015 strain S gene,compared with CH/Sichuan/S27/201 strain,mutations occurred in the nucleotides.The phylogenetic analysis showed that the S gene of CHN-SC2015 strain was close to that of the gene of the PDCoV strains,which were isolated from the mainland China.The gene was relatively close to the CH/Sichuan/S27/2012 strain and CHN-HB-2014 strain and far from the evolution of American and Korean strains.The analysis of the predicted amino acid sequence of the S protein of the CHN-SC2015 strain showed that the protein was a stable hydrophilic protein with a glycosylation site.The secondary structure prediction showed that there was abundant a-helix in the protein and the transmembrane region prediction of this protein presented there was a transmembrane region in it.2.Expression and identification of PDCoV S1 proteinThe partial S gene fragment(106-1290 bp)was chosen according to B-cell linear epitope prediction,and then sub-cloned into pET-22b(+)expression vector.The recombinant protein was 46 kDa in weight.The optimal expression condition was inducing the Transsetta strain under the final concentration of IPTG 0.8 mM 5 h on 30 °C.The infusion analysis showed that the recombinant protein was expressed in both supernatant and inclusion body.After Ni + affinity chromatography method and the ultrafiltration tube concentration,the 5.38 mg/mL purified protein was obtained.3.Construction of anti-S1 protein monoclonal antibodyBALB/c mice were immunized with 100 ?g of recombinant S1 protein as antigen for three times.A hybridoma cell line 4G11,which secreted the monoclonal antibody with specific anti-PDCoV S protein,was screened and obtained by hybridoma technique.The subtype of monoclonal antibody was IgG 2b type.The ascites was purified by octanoic acid-ammonium sulfate precipitation method.A 25 kDa and a 50 kDa of weight proteins were showed in the result of SDS-PAGE.The concentration of monoclonal antibody before and after purification was 5.02 mg/mL and 0.842mg/mL respectively,the recovery rate was 16.77%.The titer of the purified antibody was determined by indirect ELISA at a level of above 105.Western-Blot showed that monoclonal antibody reacted well with both recombinant S1 protein and PDCoV CHN-SC2015 strain.The results of indirect ELISA showed that the monoclonal antibody was negatively correlated with PEDV,Japanese encephalitis Virus(JEV),Porcine reproductive and respiratory syndrome virus(PRRSV),Classic swine fever virus(CSFV),Porcine Circovirus(PCV),Foot and mouth disease virus(FMDV)and Pseudorabies virus(PRV).In this study,the PDCoV S gene was successfully amplified and the variability analysis was carried out.The S1 protein was expressed and anti-S1 monoclonal antibody was prepared,which provided the basis for the subsequent research on the diagnosis and prevention of PDCoV. |
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