作　　者： ;

机构地区： 呼伦贝尔学院

出　　处： 《内蒙古农业大学学报（自然科学版）》 2013年第2期96-99,共4页

摘　　要： 本研究通过在vero细胞中增殖新城疫病毒、Trizol法提取病毒RNA,应用RT-PCR法扩增NP、P和L基因,再分别克隆到pGEM-T easy载体上,经限制性内切酶酶切鉴定、PCR检测和测序确认后,分别亚克隆在真核表达载体pcDNA3.1(+)上,分别命名为pcDNA3.1(+)-NP,pcDNA3.1(+)-P和pcDNA3.1(+)-L。重组质粒pcD-NA3.1(+)-NP单独转染BHK-21细胞。经Western blot检测,认定NP基因在BHK-21细胞中成功表达。新城疫病毒NP、P和L基因的成功克隆,为建立新城疫病毒反向遗传操作系统以及实验教学和研究奠定了基础。 In this study,NDV was grown in sensitive Vero ce1ls,Vira1 RNA was extracted from the infected cells using Trizol.The extracted RNA was subjected to RT-PCR with virus-specific primer pairs,the NP,P and L gene were cloned in the pGEM-T easy vector.After identification by restriction digestion,PCR amplification and sequencing analyses,The fragments of NP,P and L from pGEM-T easy vector were subcloned into pcDNA3.1（＋） expression vector respectively and named with pcDNA3.1（＋）-NP,pcDNA3.1（＋）-P and pcDNA3.1（＋）-L.Then the recombinant vector pcDNA3.1（＋）-NP was transfected into BHK-21 cell.The identification by Western blot showed that the NP gene was successfully expressed in BHK-21 cell.This research will helpful for further study of NDV reverse-genetic system.

关 键 词： 新城疫病毒 和 基因 表达质粒

领　　域： [生物学]