人ACE2基因真核表达载体的构建及其转染人内皮细胞的研究

Construction of recombinant eukaryotic expression plasmid containing human ACE2 gene and its transfection in human endothelia cells

目的:克隆人血管紧张素转换酶2(ACE2)基因,构建其真核表达载体并将之转染入体外培养的人血管内皮细胞中。方法:采用PCR方法从人胎儿心脏cDNA文库扩增出人ACE2cDNA全长基因,克隆到pcDNA3.1-D/V5-His表达载体上,构建重组真核表达质粒phACE2,经酶切和测序鉴定。采用脂质体法将phACE2转染至人血管内皮细胞中,分别利用实时定量PCR和Western印迹检测重组ACE2的mRNA和蛋白的表达情况。结果:经PCR、酶切和基因序列测定证实重组入载体的片段(2419bp)为目的基因序列,在转染的内皮细胞中发现ACE2的mRNA和蛋白的表达明显增加(P<0.01)。结论:本研究成功构建并表达了pcDNA3.1-hACE2重组真核表达载体,为该基因在高血压病防治中的功效研究奠定了基础。

Objective：To clone and construct recombinant eukaryotic expression plasmid containing human angiotensin converting enzyme 2（ACE2）and transfected it to human endothelial cells.Methods：The full-length cDNA of human ACE2 was amplified from a Human Fetal Heart cDNA Library by PCR,and cloned into pcDNA3.1-D/V5-His expression vector（phACE2）.After the identification of enzyme digestion and sequencing,phACE2 was transfected to human endothelial cells by liposome,and the mRNA and protein expression of ACE2 was determined by real-time PCR and Western blot in endothelial cells respectively.Results：The inserted fragment of recombinant vector（2419 bp）was the sequence of target gene by PCR,enzyme digestion and sequencing determination,and ACE2 mRNA,and protein expression were markedly enhanced（P〈0.01）in the transfected endothelial cells compared with controls.Conclusion：The recombinant eukaryotic expression plasmid pcDNA 3.1-hACE2 is successfully constructed and transfected in cells,which provided the basis for the study of ACE2 functions in the prevention and treatment of human essential hypertension.