Cloning And Prokaryotic Expression Of N Gene Of PRRSV Isolates From Xinjiang

Porcine reproductive and respiratory syndrome is characterized by severe reproductive failure in sows (late-term abortions, dead, weak and mummified piglets), respiratory disease and increased preweaning mortality, and has been causing tremendous economic losses to the swine industry throughout the world. The nucleoprotein is the superiority structural protein of PRRSV. It has good immunogenicity and reactinogenicity, which will be the first choice to diagnose PRRS.In this study, one pair of specific primer of ORF7 for PRRSV was designed according to the published gene sequence of PRRSV from GenBank, and N gene of XJPRRSV1/03 strain was obtained by RT-PCR method. Then, the product was directly cloned into the pMD18-T vector, named pMD18-N, and sequenced. After sequencing, the sequence of N gene of the XJPRRSV1/03 was compared with sequences of 12 other different PRRSV strains in GenBank by DNAStar software. The results showed that the homology of nucleotide sequences and deduced amino acid sequences of N gene among XJPRRSV1/03 and North American strains was 94.9-97.4% and 95.2-98.4%, respectively. It was higher than the homology between XJPRRSV1/03 and LV, which was only 67.2% and 64.5%, respectively. Furthermore, it was found that the homology was very high among XJPRRSV1/03 and S1, VR-2332, BJ-4 and FJ04 strains, but was very low among XJPRRSV1/03 and LV. The results suggested that XJPRRSV1/03 strain belongs to North American type.Subsequently, one pair of specific primer of N gene for PRRSV was designed according to the actual amplification gene sequence of PRRSV for XJPRRSV1/03 strain. The plasmid pMD18-N and plasmid pGEX-6P-1 were digested by EcoR I and Xho I, the following coupled reaction was operated. Finally the recombinant plasmid was constructed designated pGEX-6P-N. Then pGEX-6P-N was transformed into the host cell E.coli BL21 and was efficiently expressed after induction with IPTG. By SDS-PAGE, the GST-N protein was highly expressed induced by 1.2 mmol/L IPTG at 37℃for four and half an hours. The GST-N recombinant fusion protein was identified as 39kDa.Consequencely, the GST-N fusion protein was purified by the means of GST-Sepharose 4B protein purification procedure. The results showed the expression of GST-N fusion protein was 0.93mg/mL in E.coli BL21. Western blotting assays was suggested that the protein could react with the porcine polyclonal antibodies against PRRSV and the recombinant was used to generate the anti-N polyclonal antibody in rabbit with titer as high as 1:214. So this assays layed foundation of founding indirect-ELISA approach to N-specific antibody of PRRSV. It is very important to prevention and control the PRRS in Xinjiang.