Preliminary Establishment Of Indirect ELISA For Detecting Antibody To Porcine Pseudorabies Virus GB And Preparation Of Monoclonal Antibodies Against GB

Porcine pseudorabies is an acute infectious disease caused by Pseudorabies virus(PRV),The infected pig shows abortion,stillbirth,mummy fetus in pregnant sows,neurological disorders and death in newborn piglets.Since 2011,pseudorabies have been a large outbreaks in many provinces in China,which causes big economic losses to the swine industry.Therefore,the establishment of effective detection and diagnosis methods is of great significance for the control and eraducation of pig pseudorabies.In this study,the recombinant PRV gB truancated protein was successfully expressed in prokaryotic expression vector pGEX-6P-1,which includes three consecutive epitope regions,the size was about 741 bp.Monoclonal antibodies to PRV gB were prepared in mice immunized with the purified recombinant gB protein.4 hybridoma cell lines secreting monoclonal antibodies were obtained by indirect immunofluorescence assay.1.Prokaryotic expression of partial gB gene of pseudorabies virusIn order to obtain an immunogen for the preparation of PRV monoclonal antibodies,three consecutive epitopes of the gB gene were amplified and cloned into the vector pGEX-6P-1 to construct the prokaryotic expression vector pGEX-6P-1-gB.Then the plasmid was transformed into BL21 competent cells and induced expression by IPTG SDS-PAGE analysis revealed that the PRV gB recombinant protein was expressed in the supernatant.The pure PRV gB recombinant protein was obtained using a GST agarose gel column.This PRV gB recombinant protein provides an antigen for subsequent immunization of BALB/c mice and the development of monoclonal antibodies against PRV gB protein.2.Preliminary establishment of indirect ELISA for antibody detectionThe purified PRV gB protein was used as the coating antigen in the ELISA.All the factors in the ELISA were optimized.The best results could be made with a coating concentration of 0.313?g/mL,1:400 dilution of serum,5%skim milk in blocking solution,blocking for 2h,and incubation 1h for primary antibody and 45 min for HRP-conjuated antibody,15 min for color development.The method could detected the antibody only to PRV,but not to PRRSV,PEDV,CSFV,which indicated that the method was specificity.The cut-off value for the ELISA was OD450nm=0.273 with 18 negative sera tested.It could be judged positive when OD450nm?0.31,and negative when OD450nm?0.273.3.Preparation of monoclonal antibodies against pseudorabies virus gB proteinIn order to develop a monoclonal antibody against PRV gB protein,SP2/0 cells were fused with spleen cells from the BALB/c mice immunized with the purified PRV gB recombinant protein.Positive hybridoma cells were screened by indirect immunofluorescence and performed twice by limiting dilution subcloning.4 strains of hybridoma cells secreting antibodies to PRV gB protein were obtained,and named PRV gB-3H9,PRV gB-4C3,PRV gB-5F7,PRV gB-6B9,respectively.The results of Western Blot showed that only monoclonal antibody PRV gB-5F7 could specifically react with the cells infected with PRV.This monoclonal antibody subclass is IgG.The titer was 1:102400 in ELISA and 1:12800 in IFA.