猪繁殖与呼吸综合征病毒ORF5/6/7基因真核载体的构建

Construction of Eukaryotic Expressing Vector from ORF5/6/7 of Porcine Reproductive and Respiratory Syndrome Virus

针对PRRSV ORF5～ORF7设计3对特异性引物,以PRRSV GD-XH株和GD08-1株为模板进行RT-PCR扩增,并将扩增产物克隆到真核表达载体pEGFP-N1中,获得重组质粒pEGFP-N1-(ORF5～ORF7),然后将重组表达质粒转染Marc-145细胞.通过荧光显微镜观察显示,重组质粒pEGFP-N1-(ORF5～ORF7)和载体pEGFP-N1均有绿色荧光出现.结果表明:成功构建了表达PRRSV GD-XH株和PRRSV GD08-1株的GP5、M、N蛋白的真核表达质粒pEGFP-N1-GD-XH-GP、pEGFP-N1-GD-XH-M、pEGFP-N1-GD-XH-N、pEGFP-N1-GD08-1-GP5、pEGFP-N1-GD08-1-M、pEGFP-N1-GD08-1-N.

Specific primers aiming to ORF5-ORF7 of porcine reproductive and respiratory syndrome（PRRSV） were designed.The target segments with restricted enzyme digestive sites were obtained by RT-PCR with PRRSV GD-XH and GD08-1 as template.All the segments were inserted into vector pEGFP-N1 to obtain six recombinant plasmids：pEGFP-N1-GD-XH-GP5,pEGFP-N1-GD-XH-M,pEGFP-N1-GD-XH-N,pEGFP-N1-GD08-1-GP5,pEGFP-N1-GD08-1-M,pEGFP-N1-GD08-1-N.The recombinant plasmid was transfected into Marc-145 cell.Fluorescence microscopy showed that EGFP were expressed in all transfected cells.