Prokaryotic Expression Of Major Epitope Domain Of Porcine Pseudorabiesvirus Structural Protein GB And GD And The Establishment Of Indirect ELISA Against GB Antibody Of PRV In Pig Serum

Pseudorabies virus(PRV), is a member of Alphaherpesvirinae, herpesviridae, which can cause Aujeszky's disease in swine, bovine, sheep and wild animals. It is essensial to develop a specific and sensitive method to help the epidemiology survey of PRV in our country. The PRV structural protein gB and gD are much more preserved. They are major immunogenicity proteins that can be used as perfect diagnostic antigen. In this reaserch, the genes encoding the major epitope domain of gB and gD were expressed in Escherichia coli expression system respectively, then the purified recombinant gB and gD protein were obtained. The gB-ELISA was developed by using the purified recombinant gB protein as antigen. The main study were summarized as following:1. Prokaryotic Expression of Major Epitope Domain of Porcine Pseudorabies Virus Structural Protein gB and gD.According to the published sequence of the PRV china strain, the genes encoding the mahor epitope domain of gBand gD were amplified by PCR using synthetic oligonucleode primers. The expected 600bp and 677bp fragments were amplified and cloned into pGEM-T-Vector separately, then subcloned into the downstream of T7 promoter of an expression plasmid, pET-32(a). After induced by IPTG, the fusion proteins were highly expressed in Escherichia coli BL21(DE3). The expression products were about 42.4 KDa proteins. The recombinant fusion protein gB and gD occupied 60.5% and 52.3% To the total bacterial protein respectively. The purified fusion protein could both react with the pig serm conteining antibody against PRV, that is identified by SDS-PAGE and Western-blot. The expression products were purified from bacterial inclusion bodies by His·Bind affinity chromatography and the concentration of purified expression product was 1mg/ml and 0.98mg/ml respectively2 The extablishment of indirect gB-ELISAThe indirect gB-ELISA was extablished by using the recombinant fusion protein gB as antigen. The reaction condition of the gB-ELISA were explored and optimized. The optimal coating concentration was 315 ng per well, the optimal coation condion was at 4℃over night, the seum sample was diluted to 1:40,serum sample and HRP-labeled rabbit anti-porcine IgG should both be incubated at 37℃for 30 minutes. The substrate was added and incubated at 37℃for 10 minutes before terminated with stopping solution. The cutoff was determined to be 0.1×ODP+0.9ODN. When using this method to detecte samples of pig serum, the background value of negative sample is very low and to stong positive sample, the upper OD450 value can be up to 2.0 above. The results were negative when the antiserum against PCV, PPV, HCV, JEV and PRRS were detected by the optimal procdures. Results of reproducibility intra-assay shows the coefficient of variation(CV) were less than 8%. All results indicated that this indirect gB-ELISA has high specificity and good repeatability.Swine serum samples were detected by gB-ELISA and the IDEXX gB ELISA kit respectively. The sensitivity and speciticity of indirect gB-ELISA estabilished in the study were75%and 80.7%respectively to IDEXX gB ELISA. The coincidence rate between these two ELISA was79%.