天津医药

• 实验研究 • 上一篇    下一篇

Bac-to-Bac杆状病毒表达系统在BHK细胞表达鼠IL-2蛋白的研究

黄大林   

  1. 桂林医学院
  • 收稿日期:2010-09-09 修回日期:2010-11-29 出版日期:2011-02-15 发布日期:2011-02-15
  • 通讯作者: 黄大林

Bac-to-Bac baculovirus expression system in BHK cells expressing mouse IL-2 protein

  • Received:2010-09-09 Revised:2010-11-29 Published:2011-02-15 Online:2011-02-15

PDF (PC)

2581

摘要： 目的 利用Bac-to-Bac 杆状病毒表达系统在BHK细胞表达重组鼠IL-2蛋白。 方法 用Flag进行标记鼠IL-2并加入真核细胞启动子CMV，构建重组质粒pFB-CMV-IL-2并予酶切和PCR鉴定；将鼠IL-2 克隆入杆状病毒表达载体pFastBacDual中，将pFB-CMV-IL-2转化到含杆状病毒穿梭载体Bacmid的DH10Bac感受态菌中，筛选阳性克隆，获得重组杆状病毒载体Bacmid-pFB-CMV-IL-2，抽提质粒；用脂质体转染法将Bacmid-pFB-CMV-IL-2转染Sf9昆虫细胞包装病毒，收获重组杆状病毒，将构建病毒转导BHK细胞培养，用Western Blotting检测IL-2蛋白。结果 在BHK细胞成功表达重组的鼠IL-2蛋白。结论 本试验运用重组杆状病毒在真核细胞中成功表达重组鼠IL-2蛋白。

关键词: 鼠白细胞介素-2(IL-2), 杆状病毒, 载体, 质粒

Abstract: Objective The Bac-to-Bac baculovirus expression system in BHK cells expressing recombinant mouse IL-2 protein. Methods Flag to mark mouse IL-2 and join the eukaryotic cell promoter CMV, recombinant plasmid pFB-CMV-IL-2 and to the restriction enzyme digestion and PCR identification; the mouse IL-2 was cloned into the baculovirus expression vector pFastBacDual in the pFB-CMV-IL-2 was transformed into baculovirus shuttle vector containing the DH10Bac Competent Bacmid bacteria, the clones were recombinant baculovirus vector Bacmid-pFB-CMV-IL-2, extracted plasmid ; with liposome transfection method Bacmid-pFB-CMV-IL-2 transfected Sf9 insect cells packaging the virus, harvested recombinant baculovirus, will build a virus transduction of BHK cell culture, using Western Blotting Detection of IL-2 protein. Results success in BHK cells of rat recombinant IL-2 protein. Conclusion The experiment using recombinant baculovirus in mammalian cells successfully expressed recombinant mouse IL-2 protein.

Key words: mouse interleukin -2 (IL-2), baculovirus, carrier, plasmid