Prokaryotic Expression Of Porcine PRV GD And GE Genes And Development Of Monoclonal Antibodies Against PRV

Porcine pseudorabies (PR) is caused by pseudorabies virus (PRV). It is extremely contagious and acute infectious disease. The first PR case was reported in our country in 1947, and with the development of large-scale pig farming industry, more than 20 provinces and cities have reported the occurrence of the PR. This kind of disease is distributed in breeding pig farms with an extensible tendency. It results in heavy economic losses in our pig industry. There is significant value to establish its diagnosis methods for the prophylaxis and eradication of the pseudorabies.In this study, PRV JSZ strain was isolated and identified, and nucleotide distribution of the PRV was studied in the infected pigs in vivo, PRV gD and gE genes were expressed in Escherichia Coli, and the monoclonal antibodies against PRV were prepared successfully.1. Isolation and identification of PRV JSZ strain and its nucleotide distribution in the infected pigsA specific band was PCR amplified from suspicious porcine pseudorabies samples collected in a Jiangsu pig farm. The PK-15 cell produced cytopathic effect 24h postinoculation with samples. The pseudorabies virus in infected PK-15 cell was identified by PCR method and viral isolation. The PRV named as JSZ was virulent since it resulted in intense itching and death in challenged rabbits, and persistent fever and seizures in challenged piglets. The PRV nucleotide was detected widely in organs and tissues of the infected pigs by PCR method, and the highest positive ratio of samples was in spleen. In experimental challenge, the period of PRV shedding in infected pigs was about 13d when the swab samples collected from rectum or nasal cavity were detected by PCR. The isolation of PRV JSZ strain provided a biological material for further etiological research, and the study of nucleotide distribution in infected pig laid basis to detect the antigen of PRV.2. Clone and prokaryotic expression of the PRV gD and gE genesAccording to the nucleotide sequence of the PRV gD and gE genes, two pairs of primers were designed to amplify the gD ang gE gene fragments by PCR. The fragment of gD was 668bp, while the gE was 558bp, which encoding the main antigetic domains of PRV gE. The PCR products were cloned into the T7 promoter downstream of the expression plasmid pET-32a, respectively. Recombinant bacteria were identified by resistance screening and restraction endonuclease analysis and named as pET-32a-D and pET-32a-E. The recombinant proteins expressed in E. coli induced by IPTG were 45 kDa and 43 kDa, respectively, when they were analysed in SDS-PAGE. These recombniant preoteins provided basic materials for PRV antibody detection.3. Development of monoclonal antibodies against PRVPRV was proliferated in PK-15 cell. The harvested Cell cultures were purified by sucrose density gradient ultracentrifugation. The BALB/c mice were immunized subcutaneously with purified antigen of PRV. Spleen cells from immunized mice were fused with SP2/0-Ag-14 myeloma cells. Indirect enzyme linked immunosorbent assay (ELISA) was used to screen hybridoma cells, and limiting dilution method was performed to subclone positive hybridoma cells. Ten positive clones were obtained, designated as 2F5,2G10,3H10,4D11,6B4,6D5. All the McAbs belonged to IgM subgroup except 2F5, which was subtyped to IgG. These McAbs were specific for PRV and didn't have the cross reaction with other porcine viruses. These McAb made a foundation for further establishment of a PRV-testing method, which was specific, sensitive and convenient.