猪繁殖与呼吸综合征病毒SCQ株ORF5基因在E.coli中表达的初步研究

Expression of ORF5 gene of Porcine reproductive and respiratory sydrome virus in E.coli

根据已测定的猪繁殖与呼吸综合征病毒SCQ株序列设计1对引物,应用PCR方法从重组质粒PMD-ORF5扩增得到缺失信号肽序列的基因片段dORF5,将dORF5克隆至原核表达载体PBAD/Myc-His B上,成功地构建了重组表达质粒PBAD/Myc-His B-dORF5,转化大肠埃希菌TOP10感受态细胞,经阿拉伯糖诱导表达,SDS-PAGE可检测到分子质量约为19.4 ku的目的蛋白,经蛋白质分析软件Bandscan分析,其表达量可达17.1%。Western blotting分析结果表明,该重组蛋白可被兔抗PRRSV血清所识别,这为进一步研究GP5蛋白的结构、功能和开发新型诊断试剂盒、新型疫苗提供了理论与物质基础。

A pair of primers based on the sequence of Porcine reproductive and respiratory sydrome virus ORF5 gene were designed. The dORF5 gene deleted N - terminal signal peptide from recombinant plasmid PMD - ORF5 was amplified by PCR. The segment was cloned into expression vector PBAD/Myc - His B and constructed the recombinant plasmid PBAD -dORF5. The PBAD -dORF5 was transformed into E. coli TOP10 cells and the cells were induced by L- arabrose. The SDS - PAGE test indicated that the GP5 protein was expressed in E. coli cell. The molecular weight was about 19.4 ku and it can amount to 17. 1% of the total mass of bacterial proteins. The Western - blotting test showed that the protein had immunolog^cally reactive activity. This study provided fundamental data and materials for the further study on the structure and function of GP5 protein, as well as new type molecular diagnostic reagent.