中文摘要：

针对PRRSV E基因设计1对带有EcoRⅠ和SalⅠ酶切位点的引物,以PRRSV CC-1株为模版进行RT-PCR扩增,获得带有酶切位点的目的片段插入pMD18-T载体,对阳性重组质粒进行测序鉴定。将阳性质粒进行EcoRⅠ、SalⅠ双酶切,回收目的片段将其插入EcoRⅠ、SalⅠ双酶切的真核表达载体pEGFP-N1中构建重组质粒pEGFP-N1-E,将重组质粒用脂质体转染Marc-145细胞,通过荧光显微镜观察显示,在转染后24h出现荧光,48h出现荧光高峰,筛选阳性细胞株,进行目的基因转录、Western blot检测目的蛋白的表达鉴定。结果表明：成功构建真核表达载体pEGFP-N1-E,建立了稳定表达的细胞株。为研究E蛋白如何与宿主细胞结合形成通道,PRRSV吸附、穿入宿主细胞的作用机理以及筛选特效的粒子通道阻断剂奠定了基础。

英文摘要：

Specific primers containing EcoR I and Sal ~ sites and aiming to E gene of PRRSV were deigend. The objective segments with restricted enzyme dugestive sites were obtained by RT-PCR with PRRSV CC-1 as template. All the segments were inserted to pMD18-T vector,and plasmids were etracted and sequenced. The correctly inserted plasmids were were digested by EcoR I and Sal I , and the objective segments were recovered and inserted into vector pEGFP-N1, which is aslo digested by EcoR I and Sal I , to construct recombinant plasmid pEGFP-N1-E. The recombinant plasmid was transfected into Marc-145 cells by liposome-mediated, the fluorescence appears at 24 h after transfection,fluorescence peak was observed at 48 h under the fluorescent microscope. The transfected cell strain was screened to detect the target gene transcription and the expression of the target protein. The results showed that pEGFP-N1-E eukaryotic expression vector was successful- ly constructed,a stably expressing of Marc-145-E cell line was built.