| Porcine reproductive and respiratory syndrome (PRRS), characterized by severe reproductive failure in sow and respiratory disease in young pigs, was first recognized in the US in 1987. Since its appearance, PRRS has been causing tremendous economic losses to the swine industry throughout the world. 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 virulence 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, strict biosecuriry measures with the aid of intensive surveillance appear of crucial importance to protect the unaffected herds. In this study, the nucleocapsid protein gene of North American PRRSV strain VR 2332 was successfully expressed in insect cells and Escherichia Coli in soluble form respectively, and the recombinant nucleocapsid proteins were used to develop an indirect ELISA for the detection of antibodies against PRRSV. In addition, the apoptosis mechanism of Marc 145 cell infected with PRRSV was also investigated.1. Expression of porcine reproductive and respiratory syndrome virus nucleocapsid protein gene in Sf9 cell with Bac-to-Bac baculovirus expression systemNucleocapsid protein gene of PRRSV, amplified by reverse transcription-PCR, was cloned into pGEM-T Easy Vector, and subcloned into pFastfiacl donor plasmid, then inserted into baculovirus shuttle vector Bacmid based on site-specific transposition. The recombinant baculovirus, designated as reBac-N, was obtained after the recombinant shuttle vector Bacmid-ORF7 was transfected into Sf9 cells. In order to initiate the expression of nucleocapsid protein gene, a viral stock with high-titer up to 5 X 108 pfu/ml was prepared when determined by plaque assay. Infected Sf9 cells withreBac-N at an multiplicity of infection (MOI) of 5 were harvested at 72h postinfection which was verified to be the optimal time for maximum expression, and the recombinant nucleocapsid protein with molecular weight of 1 SkDa, the same as the size of nature protein, was identified to be expressed in cytoplasm in soluble, non-secreted form. Furthermore, the expression product could react with pig serum containing antibodies against PRRSV.2. Expression of porcine reproductive and respiratory syndrome virus nucleocapsid protein gene in soluble form in Escherichia coli and purification of the expression productNucleoprotein gene of PRRSV, amplified by reverse transcription-PCR, was cloned into pGEM-T Easy Vector, and subcloned into expression plasmid pGEX-6p-l. The nucleocapsid protein (N) fused to glutathione S-transfease (GST) protein was successfully expressed in soluble form in Escherichia coli BL21 after the critical conditions had been optimized. The expression product consisted of 4 protein bands of 39.5 kDa, 33 kDa, 30.5 kDa and 27.5 kDa by SDS-PAGE and western blot analysis, in which the 39.SkDa full-size fusion protein and its 30.5 kDa proteolytic fragment occupied 30.9% and 15.3% of total bacterial protein respectively. Moreover, the recombinant fusion protein GST-N could be easily purified from bacterial lysate by affinity chromatography with Glutathione Sepharose 4B, and the concentration of purified expression product was as high as 8 mg/ml. In addition, the recombinant nucleocapsid protein (N) was successfully isolated from GST-N fusion protein by cleaving between the GST moiety and the co-expressed N protein with PreScission protease. In this presentation, the recombinant fusion protein GST-N and its cleaved N protein portion showed reactivity with pig serum containing antibodies against PRRSV, it indicated that the recombinant fusion protein could be used as antigen in diagnostic assays for the detection of antibodies...
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