Preparation Of Monoclonal Antibody To GB Protein Of Porcine Pseudorabies Virus HN-1201 Strain And Identification Of B Cell Linear Epitope

Pseudorabies virus(PRV)belongs to the herpesvirus family,a-herpesvirus subfamily,and double-stranded DNA viruses.It can cause disease in pigs and a variety of domestic animals.Pigs are the main host and source of infection of the pathogen.PRV can cause reproductive disorders,fever,and acute death of piglets in sows.In recent years,swine pseudorabies has broken out in many places in my country,which has brought huge economic losses to the pig industry.The PRV genome encodes more than 70 alpha-herpes virus homologous proteins.There are 11 envelope proteins that play a role in all stages of virus replication.Among them,gB protein is the main protective antigen glycoprotein of PRV envelope,which can not only induce neutralization Antibodies are necessary for the process of virus infection and replication.This study aims to obtain PRV gB-specific monoclonal antibodies and establish a rapid detection method.In the test,7-week-old BALB/c mice were immunized with the purified PRV HN-1201 epidemic strain,and the spleen cells of the mice with positive serum antibody levels were fused with SP/20 cells.The peroxidase monolayer cell test(Immunoperoxidase Monolayer assay,IPMA),Western blot and indirect immunofluorescence assay(Indirect Immunofluorescence Assay,I FA),after subcloning screening,a monoclonal cell line 3B-3 that stably secretes PRV gB antibody was obtained.The antibody secreted by this cell line is of IgG1 subtype.The IFA titer of the antibody purified from ascites fluid is 2-11.The best dilution ratios of the cell culture supernatant and ascites purified antibody for Western blot are 1:50 and 1:5000,respectively.Hybridoma cells induce the neutralizing effect of mouse ascites.The price is 1:25.In order to identify the epitope recognized by the monoclonal antibody,a eukaryotic expression vector of gB truncated protein was constructed and transfected into cells.The truncated protein was analyzed and identified by Western blot.It was finally determined that the epitope recognized by the monoclonal antibody was 721TGLLDYSEIQRRNQLH736.Alignment with other reports of PRV gB sequence indicated that this epitope is very conserved among PRV strains.This study successfully prepared monoclonal antibodies,which provides an effective tool for the detection of PRV gB antigen and the study of the structure and function of gB protein.