【作者】 康艳梅； 赵明秋； 沈海燕； 琚春梅； 陈金顶；

【Author】 KANG Yan-Mei,ZHAO Ming-Qiu,SHEN Hai-Yan,JU Chun-Mei,CHEN Jin-Ding (College of Veterinary Medicine,South China Agricultural University,Guangdong,510642,China)

【机构】 华南农业大学兽医学院；

【摘要】 本研究针对PRRSV ORF5-ORF7设计三对带有EcoRⅠ和XhoⅠ酶切位点的引物,以PRRSV GD-XH株和GD08-1株为模板进行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、XhoⅠ双酶切,回收目的片段将其插入EcoRⅠ、XhoⅠ双酶切的真核表达载体pEGFP-N1中构建重组质粒pEGFP-N1-(ORF5～7),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,为进一步研究PRRSV编码蛋白与细胞中NF- B信号通路的关系奠定基础。更多还原

【Abstract】 Specific primers containing EcoRⅠand XhoⅠsites and aiming to ORF5-ORF7 of PRRSV were designed.The objective 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 to pMD18-T vector,and plasmids were extracted and conducted sequenceing.The correctly inserted plasmids were digested by EcoRⅠand XhoⅠ,and the objective segments were recovered and inserted into vector pEGFP-N1,which is also digested by EcoRⅠand XhoⅠ,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.更多还原