猪繁殖与呼吸综合征病毒N蛋白的表达与纯化

根据GenBank报道的PRRSV VR2332基因序列设计引物,经RT - PCR扩增,得到大小约为390 bp的阳性产物；利用Bam H Ⅰ,EcoR Ⅰ位点将N蛋白基因片段克隆到pET-28a载体,构建原核重组表达质粒pET-N,转化BL21(DE3)并进行SDS-PAGE,Western blot分析.结果表明:克隆的N蛋白基因与GenBank报道的VR2332基因同源性为93.83％；重组菌株经IPTG诱导后,N蛋白基因得到了高效表达；经筛选得到最佳诱导条件,即1 mmol/L IPTG诱导6h,蛋白表达量可达菌体蛋白总量的52.635％,SDS-PAGE后切胶回收可得到纯化的N蛋白,为进一步制备免疫胶体金试纸条或ELISA试剂盒提供基础.

Expression and Purification of Nucleocapsid Protein of Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)

On the basis of the PRRSV VR2332 sequences reported by GenBank,ORF7 gene of porcine reproductive and respiratory syndrome virus(PRRSV) were obtained by RT-PCR after designing one pair of primers and the cDNA fragment was 390 bp approximately.cDNA fragment was cloned into the multiple cloning site(BamH Ⅰ&EcoR Ⅰ) of pET28a(+) vectort and then transformed into E.coli BL21(DE3).The results were tested by SDS-PAGE and Western blot.The experimental results demonstrated that the cDNA sequence had 93.83 % homology compared to VR2332’s.The optimum condition of recombinant strain was induced by 1 mmol/L IPTG in 6 h and accumulation of the N protein was about 52.635 % of total cellular protein.The N protein which has been purified after SDS-PAGE will be used for the further preparation of immunocolloidal gold test strip or the provision of basic ELISA Kit.