重组载体pEGFP-ANG的构建及ANG基因在HUVEC中的表达

目的 :建立血管生成素 (angiogenin ,ANG)基因的真核表达系统。方法 :采用化学合成拼接法 ,获得人ANG全长cDNA并测序 ,构建重组荧光真核细胞表达质粒 pEGFP ANG ,然后经脂质体介导转染人脐静脉内皮细胞 (HUVEC)。应用荧光显微镜观测及免疫组化染色 ,对转染细胞内 pEGFP ANG的表达进行鉴定。结果 :测序表明 ,编码的氨基酸序列与人ANG完全一致。荧光显微镜下可见转染的HUVEC内发出强绿色荧光。免疫组化检测显示 ,转染的HUVEC中有棕色颗粒。结论 :获得了人ANG的全长编码基因。构建的重组体pEGFP ANG转染HUVEC后 ,可表达人ANG蛋白

Construction of recombinant vector pEGFP-ANG and expression of angiogenin gene in HUVECs

AIM: To express the recombinant angiogenin protein in mammalian cells. METHODS: Human ANG full-length cDNA was obtained by chemical synthesis. The target gene ANG was inserted into eukaryotic expression vector pEGFP-C2. The recombinant plasmid was transfected into the human umbilical vein endothelial cells (HUVECs) via Lipofectin transfection. The expression of ANG gene in transfected HUVECs was detected by fluorescence microscopy and immunohistochemical staining. RESULTS: DNA sequencing showed the sequence of the synthetic ANG was correct and amino acid sequence of ANG was right. The green fluorescence could be seen in transfected HUVECs under fluorescence microscope. Immunohistochemical staining detection showed that ANG expressed in transfected cells. CONCLUSION: Human ANG full-length cDNA has been obtained. The ANG protein was expressed in mammalian cells succesfully.