›› 2011, Vol. 23 ›› Issue (5): 330-334.doi: 10.3969/j.issn.1004-616x.2011.05.002

• 论著 • 上一篇    下一篇


熊 伟,陈贵元,何 敏,马明星,余 敏,谭德勇,*   

  1. 1. 云南大学生命科学学院,云南 昆明 650091;2. 大理学院基础医学院,云南 大理 671000
  • 收稿日期:2010-12-31 修回日期:2011-05-19 出版日期:2011-09-30 发布日期:2011-09-30
  • 通讯作者: 谭德勇

Construction of eukaryotic expression vectors of cyclin D1 and cyclin B1 and transfection into HeLa cells

XIONG Wei,CHEN Gui-yuan,HE Min,MA Ming-xing,YU Min,TAN De-yong,*   

  1. 1. College of Life Sciences, Yunnan University, Kunming 650091; 2. College of Basic Medicine, Dali University, Dali 671000, Yunnan, China
  • Received:2010-12-31 Revised:2011-05-19 Online:2011-09-30 Published:2011-09-30
  • Contact: TAN De-yong

摘要: 目的: 构建人细胞周期素cyclin D1和cyclin B1的真核表达载体,并将其瞬时转染到HeLa细胞株中。方法:以HeLa细胞总RNA为模板,通过RT-PCR扩增cyclin D1和cyclin B1基因编码的cDNA,并将扩增的cDNA片段插入p3XFLAG-CMVTM-14真核表达载体,分别构建p3XFLAG-cyclin D1和p3XFLAG-cyclin B1重组质粒,重组子经酶切分析和测序鉴定后,用脂质体介导的基因瞬时转染法,将重组正确的表达载体转染HeLa细胞,用Western-blot技术检测细胞中FLAG融合蛋白的表达。结果:经酶切鉴定和Western印迹分析证实人cyclin D1和cyclin B1的真核表达载体构建成功,并能在瞬时转染的HeLa细胞中表达分子量大小相符的重组蛋白。结论:成功构建了人cyclin D1和cyclin B1的真核表达载体,为两种细胞周期素及其相关蛋白的功能研究奠定了基础。

关键词: 细胞周期素, 载体构建, 瞬时转染, 真核表达

Abstract: OBJECTIVE: To construct eukaryotic expression vectors with FLAG epitope that can highly express human cyclin D1 and cyclin B1 genes,then transiently transfect into human cervical cancer HeLa cell line. METHODS:cDNA of cyclin D1 and cyclin B1 genes from total RNA isolated from HeLa cells was amplified by RT-PCR. After sequencing,the open reading frame (ORF) of cyclin D1 and cyclin B1 cDNA was cloned into eukatyotic expression vector p3XFLAG-CMVTM-14 to form the recombinant plasmid named as p3XFLAG-cyclin D1 and p3XFLAG-cyclin B1. Then eukatyotic expression vectors were transfected into HeLa cells by Lipofectamine 2000. Expressions of human cyclin D1 and cyclin B1 in HeLa cells were measured by Western blot. RESULTS:The result of restriction enzyme digestion and nucleotide sequencing confirmed that the recombinant plasmids were correct. And we found that the human cyclin D1 and cyclin B1 fusion protein,with the right molecular weight,was expressed in transiently transfected HeLa cells. CONCLUSION:Recombinant cyclin D1 and cyclin B1 were successfully expressed,which laid foundation for further studies of these and their related proteins.

Key words: cyclins, vector construction, transient transfection, eukaryotic expression