Expression Of Pseudorabies Virus GE Gene In Insect Cells And Establishment Of Blocking Elisa Assay For GE Antibody To Pseudorabies

Pseudorabies is one of the major infectious diseases of swine worldwide,characterized by the infertility of boars,abortion and stillbirth of sows,high mortality of sucking piglets,growth arrest of finishing pigs.In recent years,the pseudorabies shows an outbreak trend in China and results in huge economic losses to our swine industry.The pseudorabies virus(PRV)has eleven glycoprotein genes,and gE gene is one important virulence-associated gene.At present,most of commercial PRV attenuated and inactivated vaccines were based on the strains with gE gene deletion.However,vaccines could not eradicate PRV because of the latent infection of this disease.Thus,eliminating the individuals infected field pseudorabies viruses by differentiating field PRV strains infection and gE gene-deletion vaccine immunization is the fundamental measure for eradicating pseudorabies of swine.Currently,The differential diagnosis kits of PRV mainly rely on foreign expensive kit in our country,it is necessary to develop independent and excellent diagnostic products.In the study,PRV gE gene was cloned into p Fast Bac/HBM-TOPO vector,and transformed into DH10 Bac TME.coli containing baculovirus shuttle plasmid.Recombinant bacmid containing PRV gE gene was gained by three antibiotics and blue/white selection.Then recombinant Bacmid was transfected into Sf9 insect cells,and recombinant baculovirus was obtained.The gE protein expressed by Sf9 cell infected with recombinant baculovirus was identified by indirect immunofluorescence(IFA),SDS-PAGE and Western blot.The results showed that gE protein has been successfully expressed in a baculovirus expression system,and has a good reactionogenicity.6 to 8 weeks-old BALB/c mice were immunized with PRV purified by sucrose density gradient centrifugation.After four immunization,the spleen cells of the immunized BALB/c mice were fused with the myeloma cells Sp2/0.Three positivehybridomas(1H1,7D4,and 5F2)were obtained after screening by indirect ELISA based on gE antigen.Subsequently,the relative affinities and IFA titers of three monoclonal antibodies(Mc Abs)were determined.The Mc Ab 1H1 had the highest relative affinity and IFA titer of 1:1600.Biological characterization of 1H1 Mc Ab showed that the chromosome numbers of 1H1 hybridoma cells is normal,and the heavy and light chains of 1H1 Mc Ab were of Ig G1 and ? isotype.In addition,IFA test showed that the Mc Ab 1H1 had high specificity.Horseradish peroxidase(HRP)was linked to Mc Ab 1H1 by improved sodium periodate method,and then PRV gE blockingELISA was developed based on the Mc Ab HRP-1H1 and gE antigen.The BlockingELISA was used for detecting CSFV,PPV,PRRSV,JEV positive sera.All the positive sera could not be detected by this method,which indicates that the method had a good specificity.Contrast with sensitivity of IDEXX kit showed that this method has a good sensitivity.238 field serum samples were detected by this method,and the results showed that the overall coincidence rate between our method and IDEXX kits was 90.34%.In summary,the blockingELISA method developed in this study can be used for the differention between field PRV infection and gE gene-deleted vaccine immunization,which laid the foundation for the development of excellent PRV gE-ELISA diagnostic kit.