pGC-silencer-U6/Neo/GFP/ABCG2真核表达质粒的构建及鉴定  

Construction and identification of eukaryotic plasmid pGC-silencer-U6/Neo/GFP/ABCG2

作　　者：俞艳萍[1] 张松[1] 孔维佳[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院耳鼻咽喉科,武汉430022

出　　处：《临床耳鼻咽喉头颈外科杂志》2010年第17期795-798,共4页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

摘　　要：目的:构建靶向人ABCG2基因的pGC-silencer-U6/Neo/GFP/ABCG2真核表达质粒,运用RNA干扰下调ABCG2基因在鼻咽癌细胞系CNE-2中的表达,并筛选出其基因沉默效果最明显的shRNA质粒表达载体。方法:针对ABCG2基因的mRNA序列设计3个干扰靶点,分别构建3个shRNA质粒表达载体和1个阴性对照质粒表达载体,经大肠杆菌扩增,酶切、PCR、测序鉴定,用脂质体法转染鼻咽癌CNE-2细胞,用real-timePCR和Westernblot检测ABCG2基因在mRNA和蛋白水平的抑制效果。结果:经测序证实,成功构建pGC-silencer-U6/Neo/GFP/ABCG2真核表达质粒。重组质粒转染CNE-2细胞后,ABCG2基因的mRNA水平及蛋白表达水平明显下调,其中以1号重组质粒效应最强,mRNA水平下调75%,蛋白水平下调68%。结论:成功构建以人ABCG2为靶向的pGC-silencer-U6/Neo/GFP/ABCG2的重组质粒。其对鼻咽癌CNE-2细胞中AB-CG2的表达具有显著抑制效应,为进一步研究ABCG2在CNE-2细胞中的功能和鼻咽癌的基因治疗奠定了基础。Objective：To construct three short hairpin RNA（shRNA）interference expression plasmid vectors of human ABCG2 gene,to assay the expression of ABCG2 in a human nasopharyngeal carcinoma（NPC）cell line,CEN-2 cell line,and to detect the RNAi effect of shRNA.Method：Targeting ABCG2 gene sequence,three plasmid expression vectors coding for shRNA and a control vector containing random DNA fragment were constructed.The recombinant plasmids were amplified in Ecoli.DH5 and then identified by restriction digestion,PCR and sequencing.The recombinant plasmids were transfected into CEN-2 cells.ABCG2 expression was assayed by real-time quantitative PCR and Western blot.Result：The construction of pGC-silencer-U6/Neo/GFP/ABCG2 was succeed.The shRNA plasmids significantly down-regulated the ABCG2 expression in CEN-2 cells,at both mRNA level and protein level.Recombinant plasmid 1 had the strongest effect compared with plasmids 2 and 3（P0.05）,with an inhibition ratio of 75% at the mRNA level and 68% at the protein level.Conclusion：pGC-silencer-U6/Neo/GFP/ABCG2 has been successfully constructed and it can down-regulate ABCG2 expression after transfected into CEN-2 cells,which could help further studies of ABCG2 functions CEN-2 cell line and contribute to the NPC gene therapy.

关 键 词：ABCG2 鼻咽肿瘤 RNA干扰 shRNA 真核表达质粒 

分 类 号：R765[医药卫生—耳鼻咽喉科]