摘要
参照NCBI公布的施马伦贝格病毒(SBV)的N基因开放阅读框序列,设计了一对含特异性酶切位点的引物,扩增SBV N基因,将其克隆于昆虫杆状病毒表达载体p Fast Bac HTB,然后以该重组质粒转化DH10Bac感受态细胞,得到重组穿梭质粒Bacmid-SBV-N,将该重组穿梭质粒在脂质体介导下转染Sf 9昆虫细胞,得到表达SBV重组N蛋白的杆状病毒。通过SDS-PAGE和Western blot对重组N蛋白进行鉴定,表明该蛋白得到表达。本研究为以SBV核蛋白为基础的相关检测方法的建立提供了物质基础。
According to the published genome sequences of Schmallenberg virus in NCBI database,a pair of specific primers were designed to amplify the nucleoprotein（NP）of SBV gene by PCR.The amplified fragment was inserted into the baculovirus expression vector p Fast Bac HTB.After sequencing and analysis,the recombinant plasmid was transformed into E.coli DH10 Bac.Recombinant baculovirus was achieved by transfection of the Sf9 cell with BacmidN.The recombinant virus was confirmed via SDS-PAGE and western blotting,showing that N protein was expressed in infected insect cells.The expression of Schmallenberg virus N protein provided the material base to develop SBV nucleoprotein-based assays.
出处
《中国动物检疫》
CAS
2016年第1期76-80,共5页
China Animal Health Inspection
基金
国家质检总局科技项目(2013IK050)
国家科技支撑计划项目(2013BAD12B03)
