Expression Of Nucleocapsid Protein Gene Of Porcine Reproductive And Respiratory Syndrome Virus And Its Characterization

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. 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 biosecurity measures with the aid of intensive surveillance appear of crucial importance to protect the unaffected herds. In this study, the target gene of PRRSV strain YC/2007 was successfully expressed in Escherichia Coli and insect cells.1.Expression of porcine reproductive and respiratory syndrome virus nucleocapsid protein gene in Escherichia coliNucleocapsid protein gene of PRRSV, amplified by RT-PCR, was cloned into pGEM-T Easy Vector, and subcloned into expression plasmid pGEX-6p-1. The nucleocapsid protein (N) fused to glutathione S-transfease (GST) protein was successfully expressed in Escherichia coli BL21, which was identified by SDS-PAGE and Western blot with anti-N monoclonal antibody 6G7 , and the expression product consisted of 2 protein bands, one was a full-size fusion protein band of 39.5kDa, the other was a proteolytic fragment of 30.5kDa.2.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 was cloned into pFastBac1 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. Nucleocapsid protein was expressed in the Sf9 cells at 72h post-infection with reBac-N, which was identified by immunofluorecent assay and western blot with anti-N monoclonal antibody 6G7, and the recombinant nucleocapsid protein with molecular weight of 15kDa, the same as the size of nature protein.