Prokaryotic Expression of ORF7 Gene Fragment of North American Genotype Porcine reproductive and respiratory syndrome virus and the Immunogenicity Analysis of the Expressed Protein
Abstract：Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major pathogens causing reproductive and respiratory symptoms in pigs (Sus scrofa), and N protein is the main structure protein and antigen protein of the virus. Therefore, this experiment was aimed to study the immunogenicity of N protein of North American genotype Porcine reproductive and respiratory syndrome virus (NA-PRRSV). The gene was amplified from the RNA of NA-PRRSV QH-08 strain by RT-PCR, whose product was approximately 372 bp. The product was cloned into pET30a(+)vector, RT-PCR, digestion and sequencing were used to identify positive plasmid. The identified recombined plasmid was expressed by Escherichia coli BL21 (DE3) and was induced by 1 mmol/L isopropy-β-D-thiogalactoside (IPTG) at 37 ℃. The recombinant N protein was purified by Ni-NTA and refolding. The expressed recombinant N proteins was used as an immunogen for immunization of New Zealand white rabbits (Oryctolagus cuniculus) by subcutaneous injection in several sites on back. The specificity of the serum antibody was determined by Western blot. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDA-PAGE) showed that the recombined protein was successfully expressed in E. coli BL21 (DE3) with a relative molecular weight of 24 kD. The results of Western blot showed that this recombined N protein was specifically reacted with NA-PRRSV positive pig serum; No cross reacted with Porcine parvovirus type 1 (PPV1) and PPV2 positive pig serum. The prepared lepus antisera was specifically reacted with recombined N protein. The recombinant vector pET30a-NA-PRRSV-ORF7 was successfully constructed, and the recombined N protein with excellent immunogenicity was successfully expressed in E. coli which provided a theoretical basis for the diagnosis of NA-PRRSV.
