Study On Differential Diagnostic Assays Of Pseudorabies And The Sequence Of Porcine Reproductive And Respiratory Syndrome Virus Genome

Pseudorabies virus (PrV), a member of Alpha Herpesvirus, is the causative agent of Pseudorabies (Aujeszky's disease), one of the most serious infectious diseases of several domestic and wild animals. Swine was the natural host and reservoir of PrV. Pseudorabies was an economically important disease of swine industry in China. Currently it is an important and difficult task to prevent, control and eradicate this disease in China. Use of an effective gE or gG gene-deleted PrV vaccine combined with a differential diagnostic kit for PrV glycoprotein gE or gG has proven successful in several Pseudorabies-eradication. gE and gG are unessential for viral replication in cell culture, however gE played an important role in determining virulence and neurotropism. In order to develope a PrV-gE(or gG) differential diagnostic method for the pseudorabies eradication program in China, the following researches were explored.1. The cloning of PrV gG gene and it's expression in E.coli.The complete glycoprotein gG gene of pseudoiabies virus Ea strain was amplified by PCR technique and cloned into pBluescriptIISK+. The gG gene was sequenced by sanger's sequencing technique. Then, the gG gene was inserted into downstream of the T7 promoter of an expression vector, pET-28a, to yield the recombinant plasmid pET-28a-gGi, but the expression failed. Then the plasmid-pET-28a-gGi was digested by Not I to delete the short fragment of downstream of gG gene and linked to yield the recombinant pET-28a-gG2. After induced by IPTG, a high expression of fusion protein was obtained. SDS-PAGE analysis showed that the fusion protein was 47kD and the product of expression was specific to antisera against PRV by protein dot blot analysis. The fusion protein existed mainly in form of soluble protein. After be purified by saturated (NH4)2SO4 solution, the product was specific to swine antisera against PRV in indirect ELISA.2. The development of gG-LAT and gG-ELISAThe gG latex agglutination test (LAT) and the gG Enzyme linked Immunosorbent Assay (ELISA) based on recombinant glycoprotein gG for differentiation between infected and vaccinated pigs was developed. gG-LAT can significantly differentiate the infected from vaccinated (gG deleted caccine) pigs. Statistical result of testing 340 serum samples indicated that the diagnosis method has high specificity and sensitivity. The specificity and sensitivity of the developed gG-ELISA were evaluated by testing 100 serum samples. The result of clinical application indicated that there is high coincidence rate between gG-ELISA and other clinical diagnosis methods. 842 serum samples of pigs that were vaccinated by gG- marker vaccines have been tested by gG-ELISA forepidemiologic investigation, and 242 sera of them were examined to be positive. The positive rate was 28.74%. The above results indicated that the gG-LAT and gG-ELISAcan be used for the differentiation between the pseudorabies virus (PrV) infected and vaccinated (gG' vaccine) pigs.3. The expression of the major epitope domain of glycoprotein gE of pseudorabies virus in E.coli.A 0.8kb DNA fragment encoding the major epitope domain of glycoprotein gE of pseudorabies virus Ea strain was inserted into the downstream of the T7 promoter of an expression vector, pET-28b, to yield the recombinant plasmid pETgE804. After induced by IPTG, a high level expression of fusion protein was obtained. SDS-PAGE and Western blotting analysis showed that the fusion protein was 38kD and the protein was specific to antisera against PRV Ea strain.4. The development of gE-LAT and gE-ELISAThe gE protein existed mainly in form of inclusion body. After being denatured and renatured, the protein was used to sensitize latex to prepare the latex antigen. Then, the concentration of antigen, times and temperature for sensitization were optimized. The latex agglutination test (LAT) based on recombinant glycoprotein gE for differentiation between infected and vaccinated (gE deleted vaccine) pigs was developed. The specificity and sensitivity of the developed gE-LAT were also...