Preparation And Preliminary Application Of Monoclonal Antibody Against Porcine Deltacoronavirus E Or NS7 Protein

Porcine deltacoronavirus(PDCoV)is an enteropathogenic coronavirus that causes diarrhea,vomiting,dehydration and even death in neonatal piglets.Although the fatality rate caused by PDCoV infection is lower than that of PEDV,PDCoV is frequently coinfected with PEDV,TGEV,and other viruses.This has brought a serious challenge to the prevention and control of porcine enterovirus and seriously affected the development of pig husbandry.E protein,one of the components of the PDCoV envelope,involves in the assembly and germination of the virion and can effectively induce the immune response.NS7 protein,an accessory protein with species specificity,is unnecessary for viral replication.In this study,mAbs against PDCoV E protein and mAbs against PDCoV NS7 protein were prepared.It provides materials for the functional study of PDCoV accessory protein and the establishment of relevant detection methods.The monoclonal antibodies(mAbs)against E protein were preliminary used in immunohistochemistry(IHC),which laid the foundation for the detection of PDCoV antigen in tissues.The research contents are as follows:1.Preparation of mAbs against PDCoV E proteinThe E gene was amplificated by PCR using pCAGGS-HA-E eukaryotic expression plasmid as template and then cloned into pGEX-6p-1 to obtain the recombinant plasmid pGEX-6p-1-E,which can be induced to express fusion protein.SDS-PAGE confirmed that the fusion protein GST-E was mainly expressed as inclusion bodies.The fusion protein was purified by inclusion bodies purification.The concentration of purified fusion protein was 2.0 mg/m L.Female BALB/c mice were immunized with the obtained purified protein GST-E.When the serum antibody level of mice reached fusion requirement,spleen cells were taken and fused with SP2/0 myeloma cells.After indirect ELISA screening and subclone culture,4 cell lines stably secreting mAbs against PDCoV E protein were obtained,which were named as DE-4F7,DE-8E8,DE-8F9,DE-10G3.The obtained mAbs were identified by Western Blot and IFA.These assays confirmed that all 4 mAbs specifically reacted with the PDCoV E protein of eukaryotic expression,PDCoVinfected cell,and purified PDCoV virion.Chromosome analysis showed that the obtained hybridoma cell lines had clear chromosome morphology with the number of chromosomes ranging from 90 to 110.Subtype analysis showed that 4 mAbs had κ light chains,and had heavy chains of different IgG subtypes.2.Preparation of mAbs against PDCoV NS7 proteinThe mAbs using the full length of PDCoV NS7 protein as immunization antigen were prepared in our laboratory and could reacted with NS7 a protein,a 100 amino acid(aa)polypeptide identical to the 3’ end of NS7.Therefore,in this study,the mAbs against N-terminal 100 aa of PDCoV NS7 protein was prepared.The prokaryotic expression plasmid pGEX-6p-1-NS7(1-100aa),which was preserved in our laboratory and expressed the N-terminal 100 aa of PDCoV NS7,was induced to express fusion protein.SDS-PAGE confirmed that the fusion protein GSTNS7(1-100aa)was expressed as inclusion bodies.The protein was purified by inclusion bodies purification.The concentration of purified fusion protein GST-NS7(1-100aa)was 8.8 mg/m L.The fusion protein GST-NS7(1-100aa)was used to immunize BALB/c mice.When the serum antibody level of mice reached fusion requirement,spleen cells were fused with SP2/0 myeloma cells.After indirect ELISA screening and subclone culture,5 hybridoma cell lines secreting mAbs against NS7 protein were obtained,which were named as DNS7-1E6,DNS7-2A9,DNS7-2D4,DNS7-4B6,DNS7-6A12.Western Blot and IFA assays confirmed that all 5 mAbs specifically reacted with PDCoV-infected cell,and that DNS7-1E6,DNS7-2A9 and DNS7-4B6 specifically reacted with the NS7(1-100aa)and NS7 protein of eukaryotic expression,rather than NS7 a protein of eukaryotic expression.Chromosome analysis showed that the obtained hybridoma cell lines had clear chromosome morphology and the number of chromosomes were ranging from 90 to 110.Subtype analysis showed that all 5 mAbs had light chains of κ type,and had heavy chains of IgG2b subtype.3.Preliminary Application of mAbs against PDCoV E Protein in IHCThe jejunum and ileum of piglets infected with PDCoV,PEDV or TGEV by oral administration were taken.These tissue samples were embedded in paraffion and cut in sections.Then the prepared mAbs were used as the primary antibody in these sections for IHC.The results showed that DE-10G3 mAb against E protein could specifically bind to PDCoV antigen in jejunum and ileum tissues,but could not bind to PEDV or TGEV antigen.However,all mAbs against NS7 protein could not specifically bind to PDCoV antigen in jejunum and ileum tissues.These results indicated that the DE-10G3 mAb against E protein had good specificity which could be used for immunohistochemical detection of PDCoV antigen in the intestinal tissues of piglets.