摘要
为实现番鸭呼肠孤病毒（MDRV）YB株σNS蛋白的真核表达,首先经PCR扩增了σNS基因的完整编码序列（CDS）,连接到携带有flag标签的真核表达载体pCI-neo-flag,双酶切鉴定及序列测定表明成功构建了重组表达质粒pCI-flag-σNS,使用ViaFect转染试剂将其转染至DF-1细胞,采用半定量RT-PCR法检测σNS基因的转录水平,Western-blot分析σNS蛋白的表达。RT-PCR结果显示,σNSmRNA水平在转染后0~36h逐渐递增,48h时出现下降;Western-blot鉴定结果显示,重组σNS蛋白与flag鼠单克隆抗体和MDRV-σNS兔多克隆抗体均能发生特异性反应;以上结果均表明MDRV-YBσNS基因在DF-1细胞中得到了正确的表达,为后续蛋白功能研究奠定了基础。
To expressσNS protein of Muscovy duck reovirus（MDRV）YB strain in eukaryotic cells,theσNS gene was amplified and correctly inserted into eukaryotic expression plasmid pCIneo-flag,and then transfected in DF-1cells with ViaFect transfection reagent,and transcription and expression levels ofσNSgene were detected by semi-quantitative RT-PCR and Western-blot analysis,respectively.Results showed that theσNS mRNA levels increased gradually at 0-36 hposttransfection,while with a declination at 48h;Western-blot analysis also confirmed that the expressedσNS protein could react specifically to both anti-flag mouse monoclonal antibody and antiMDRV-σNS rabbit polyclonal antibody.All above results indicated that MDRV-YBσNS was successfully expressed in DF-1cells,which would lay foundations for further functional studies.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2016年第3期419-422,484,共5页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(U1305212)
