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

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。

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

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.