猪繁殖与呼吸综合征病毒HBKM2株ORF7基因的克隆、序列分析及原核表达

Cloning,Sequence Analysis and Prokaryotic Expression of ORF7 Gene of Porcine Reproductive and Respiratory Syndrome Virus Strain-HBKM2

[目的]对猪繁殖与呼吸综合征病毒HBKM2毒株核蛋白编码基因ORF7进行研究,为进一步了解其编码蛋白的功能和研制新的血清学诊断方法奠定基础。[方法]采用RT-PCR方法扩增HBKM2株的ORF7基因,然后利用DNA Star软件包对克隆的序列进行序列分析,将ORF7基因克隆到大肠杆菌原核表达载体pGEX-KG上,构建重组表达质粒pKG-N。将重组质粒转化到大肠杆菌BL21,并经IPTG诱导表达,最后利用Western-blot对表达蛋白的免疫反应活性进行鉴定。[结果]HBKM2毒株的ORF7基因长约372 bp;序列分析表明与高致病性猪繁殖与呼吸综合征病毒的同源性达99%以上;SDS-PAGE表明克隆的ORF7基因在大肠杆菌中获得了高效融合表达;Western-blot分析表明,重组蛋白GST-N能够与PRRSV高免猪血清发生特异性的免疫反应。[结论]成功地扩增了ORF7基因,且GST-N融合蛋白能在大肠杆菌中高效表达。

[ Objective ] The foundation of the further understanding of the function of encoding protein and the development of new serological diagnostic method of porcine reproductive and respiratory syndrome virus was laid through the study on the ORF7 Gene of porcine reproductive and respiratory syndrome virus strain-HBKM2. [ Method ] The ORF7 gene of strain HBKM2 was amplified with the method of RT-PCR and then the cloned sequence was analyzed with DNA Star software. The ORF7 gene was cloned into E. coli pGEX-KG vector to construct recombinant expression plasmid pKG-N. Then the recombinant plasmid was transformed into E. coli BL21 and induced to express by IPTG. Finally the immune response activity of expressed proteins was identified with the method of Western-blot. [ Results ] The ORF7 genes of strain HBKM2 was about 372 bp. Sequence analysis showed its homology with HP-PRRSV was more than 99%. SDS-PAGE showed that the cloned ORF7 gene could be highly expressed in E. coli. Western-blot analysis showed that the recombinant protein GST-N could induce specific immune response with hyper immune serum of PRRSV. [ Conclusion ] ORF7 gene was amplified successfully and GST-N fusion protein could be expressed efficiently in E. coli.