慢病毒重组ACE2基因表达载体的构建及其感染人脐静脉内皮细胞的研究

Construction, packaging of recombinant ACE2 gene lentiviral vector and its infection in human umbilical vein endothelial cells

目的构建携带血管紧张素转换酶2（ACE2）基因的慢病毒表达载体，观察其感染人脐静脉内皮细胞后ACE2基因的转录及表达水平。方法采用PCR技术从含有ACE2基因的质粒克隆模板pc—DNA3．1-hygro（＋）一mACE2上钓取ACE2基因，并将ACE2基因重组到慢病毒载体表达质粒pGC—FU中，构建重组慢病毒载体表达质粒pGC—FU—ACE2，通过酶切、测序验证ACE2基因后，将pGC—FU—ACE2质粒和包装质粒pHelper1．0、pHelper2．0共同转染人胚胎肾上皮细胞系293T细胞，获得携带ACE2基因的重组慢病毒Lentiviral—ACE2；然后感染人脐静脉内皮细胞，通过RT—PCR、Western—blot检测ACE2基因的转录和蛋白表达水平。结果成功构建携带ACE2基因的慢病毒表达载体Lentiviral—ACE2，并获得大量高纯度的慢病毒浓缩液。目的基因ACE2能被重组慢病毒高效地导入人脐静脉血管内皮细胞，并能高水平表达。结论慢病毒表达载体Lentiviral—ACE2成功构建并能高效率感染人脐静脉内皮细胞，为研究ACE2对血管内皮细胞的影响和更深入地探索ACE2基因的功能奠定了基础。

Objective To construct, package a lentiviral vector carrying ACE2 gene and investigate its transcription and expression level in human umbilical vein endothelial cells （HUVEC）. Methods ACE2 gene was amplified from plasmid pc-DNA3.1-hygro （＋）-mACE2 by PCR technique and subcloned into the expression plasmid of pGC-FU lentiviraI vector. The pGC-FU-ACE2 Lentiviral expression vector was constucted.The correct ACE2 gene was confirmed by endoenzyme digestion and sequencing. Recombinant Lentiviral-ACE2 were produced by 293T cells following the co-transfection of pGC-FU-ACE2 and packaging plasmids-pHelper 1.0 and pHelper 2.0. The recombinant lentiviruses （Lentiviral-ACE2） were then used to infect human umbilical vein endothelial ceils. The transcription and protein expression level in human umbilical vein endothelial cells of ACE2 were detected by RT-PCR and Western blotting respectively. Results The recombinant lentiviruse Lentiviral-ACE2 was produced successfully, and the highly-purified virus liquid was obtained. Besides, it could deliver target gene ACE2 to HUVEC, and make the ACE2 express highly in HUVEC. Conclusion The recombinant lentiviruse Lentiviral-ACE2 was produced successfully and it can deliver target gene ACE2 to HUVEC efficiently, which pro- vide the basis for the further study of ACE2 functions.