中国医科大学学报

中国医科大学学报

中国医科大学学报 ›› 2012, Vol. 41 ›› Issue (6): 494–496.

• 基础医学 • 上一篇    下一篇

p53RFP 表达载体的构建及其对HEK293A细胞增殖的抑制作用

裴静娴1,王月刚1,张艺军 2,赖文岩1,吴平生1   

  1. (1.南方医科大学南方医院心内科,广州 510515;2. 广州军区广州总医院干部病房二科,广州 510010)
  • 收稿日期:2012-09-25 修回日期:2012-09-25 出版日期:2012-06-20 发布日期:2012-09-27
  • 基金资助:
    国家自然科学基金青年基金资助项目(30800462)

Construction of a Human p53RFP Expression Vector and Its Inhibitory Effects on Proliferation in HEK293A Cells

PEI Jing-xian 1,WANG Yue-gang1, ZHANG Yi-jun2, LAI Wen-yan1, WU Ping-sheng1   

  1. (1. Department of Cardiology, Nan Fang Hospital , Southern Medical University, Guangzhou 510515, China; 2. The Second Department of Cadre Ward, General Hospital of Gungzhou Military Command of PLA, Guangzhou 510010, China)
  • Received:2012-09-25 Revised:2012-09-25 Online:2012-06-20 Published:2012-09-27

摘要: 目的 构建p53RFP 表达载体,观察p53RFP 表达上调对HEK293A细胞生长的影响。方法 利用DNA克隆技术,将人p53RFP基因的编码序列克隆至质粒pcDNA 4/Myc-HisA,经限制性酶切、DNA测序鉴定重组载体。质粒扩增提取,经脂质体介导法转染HEK293A细胞,实时定量RT-PCR、Western blot检测转染后p53RFP mRNA及蛋白的表达水平。采用CCK-8检测p53RFP 表达上调对HEK293A细胞增殖的影响。结果 p53RFP转染后其mRNA及蛋白表达水平均显著上调;p53RFP转染后72 h、96 h细胞增殖受到显著抑制(P<0.01)。结论 成功构建p53RFP表达载体pcDNA 4/-p53RFP,并可在HEK293A细胞有效表达,其表达可抑制HEK293A细胞增殖。

Abstract: Objective To construct a p53RFP expression vector and examine explore the effect of highly expression of p53RFP on the proliferation of HEK293A cells. Methods The entire coding sequence of human p53RFP gene was cloned into pcDNA 4/Myc-HisA vector by means of DNA cloning technique. The product was confirmed by enzymatic digestion and sequencing. The vector was amplified and extracted, then transfected into HEK293A cells via liposome, and the levels of p53RFP mRNA level and protein level wereas detected by quantitative real-time RT-PCR and Western blot . Cell proliferation of HEK293A was measuredevaluated by CCK-8. Results The mRNA and protein levels of in p53RFP both remarkably increased remarkably after HEK293A cells was transfected with p53RFP expression vector. Cell proliferation of HEK293A was significantly inhibited at the time points of 72h and 96h after p53RFP transfection(P<0.01). Conclusion The p53RFP expression vector was successfully constructed successfully, which can be effectively transfected into HEK293A cells . The highly expression of p53RFP could inhibit the cell proliferation of in HEK293A.

Key words: p53RFP, expression vector, HEK293A

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  • 中图分类号 R3 文献标志码 A 文章编号
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