| Porcine reproductive and respiratory syndrome (PRRS) was an urgent contagion produced by porcine reproductive and respiratory syndrome virus (PRRSV), it has been broken in American in 1987 at the first time, subsequently spreaded in all over the world. Since it's appearance, PRRS has been causing tremendous economic losses to the swine industry throughout the world. PRRSV pathogen was first separated by Guo Baoqing in China, which reported in 1996. Now, this disease has become one of the major illness harmed swine breed aquatics. Although PRRSV isolates identified from around the world cause similar diseases in pigs, increasing data indicate that PRRSV strains are antigenically and genetically heterogenous and differ in virμLence in infected pigs. It is evident that the current vaccines are not effective in protecting against infections with the genetically diverse field strains of PRRSV, and the attenuated vaccine viruses can revert genetically to cause clinical disease. Up to now, vaccination is not effective to control the disease, therefore, To establish a fast and effective method towards the disease is the important base for internal swinery's investigate of epidemiology and immunity. In addition, the apoptosis mechanism of Marc145 cell infected with PRRSV was also investigated.Nucleocapsid (N) protein was the major structural protein of virus, which encoded by ORF7. It has perfect immunogenic and highly conservitive in serotype. According to the PRRSV's sequence of American-type, one pair of primer included restricts enzyme cleavage sites BamH and Xho I were designed that can amplify N gene. By RT-PCR, a gene fragment of about 372bp was amplified. And then, the fragment was cloned into pMD18-T vector. After the sequence determination was got, it was compared with sequence of GenBank using blast tool and discovered that this gene was same to the CH-1a virus. Expressed plasmid pGEX-4T-1-N was constructed The recombinant plasmid was transformed into E.coli Rosetta, and induced with IPTG for expression. Then, the fusion protein about 40 ku was expressed. The size was accord with anticipate. By analysis of Western-Blotting, the expressed production can react with antibody to PRRSV. The resμLt indicated that nucleocapsid protein of PRRSV was expressed in the prokaryote system. it indicated that the recombinant fusion protein coμLd be used as antigen in diagnostic assays for the detection of antibodies to PRRSV.The recombinant nucleocapsid protein GST-N expressed in Escherichia coli Rosetta could all be used as antigens in the development of an indirect ELISA for the detection of antibodies against PRRSV with high specificity and sensitivity, well performed, therefore, the GST-N was chosen as the reagent in the assay because it was more economical and convenient to prepare. The optimal coating concentration of antigen was determined by checkerboard titration using hyperimmune pig antiserum to PRRSV, and amounted to 1.2 mg/L of GST-N fusion protein. The serum sample for testing was diluted to 1:80. A total of 192 serum samples originating were used for validation of this recombinant nucleocapsid protein-based indirect ELISA.Compared to PRRSV as antigene ELISA kit, the sensitivity and specificity of recombinant nucleocapsid protein-based indirect ELISA established in this study were 96.5% (185/192). The data in this study demonstrated that the recombinant nucleocapsid protein-based indirect ELISA was not only reliable with high sensitivity and specificity, but also was economical and convenient in terms of antigen production. Thus, it would be a very useful tool for routine diagnostics, epidemiological surveys, and outbreak investigations. |
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