靶向人SIRT1基因的shRNA真核表达质粒的构建及鉴定  

Construction and identification of eukaryotic plasmid expression vectors encoding the short hairpin RNA targeting human SIRT1

作　　者：崔静[1] 赵刚[1] 孙仁虎[1] 勾善淼[1] 黄敏[1] 王春友[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院胰腺外科,湖北武汉430022

出　　处：《中国普通外科杂志》2010年第3期259-263,共5页China Journal of General Surgery

基　　金：国家自然科学基金资助项目(30600594)

摘　　要：目的构建针对人SIRT1基因的shRNA真核表达质粒,并筛选出对胰腺癌细胞系PANC-1基因沉默效果最明显的shRNA质粒表达载体。方法针对SIRT1基因的mRNA序列设计,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,转染胰腺癌PANC-1细胞,实时荧光定量PCR和Western blot检测SIRT1 mRNA和蛋白的被抑制情况。结果经测序证实,成功构建SIRT1-shRNA真核表达质粒,插入的DNA片段的序列与设计序列完全一致。重组质粒转染PANC-1细胞后,SIRT1 mRNA和蛋白水平明显下调;其中以1号重组质粒效应最强。结论成功构建了携带以SIRT1为靶向的shRNA的重组质粒。其对胰腺癌PANC-1细胞SIRT1的表达具有显著抑制效应。该实验为进一步研究SIRT1的功能和肿瘤的基因治疗提供了实验基础。Objective To construct three short hairpin RNA（shRNA） interference expression plasmid vectors of human SIRT1 gene,assay the expression of SIRT1 in pancreatic carcinoma PANC-1 cells after transfecting with recombinant plasmids,and detect the RNAi effect of shRNA.Methods Three plasmid expression vectors coding for shRNA targeting SIRT1 gene sequence and a control vector containing random DNA fragment were constructed.The recombinant plasmids were amplified in E.coli.DH5α was identified by restriction digestion,PCR and sequencing.The vectors were transfected into PANC-1 cells.SIRT1 expression was assayed with real-time quantitative PCR and Western blot.Results The successful construction of recombinant plasmids was confirmed by DNA sequencing of the inserted segments.Transfection of shRNA plasmids significantly down-regulated SIRT1 expression in PANC-1cells.Recombinant plasmid 1 had the strongest effect,with an inhibition ratio of 79.43% at the mRNA level and 83.27% at the protein level,which showed a significant difference from plasmids 2 and 3（P 0.05）.Conclusions Plasmid vector expressing shRNA against SIRT1 has been successfully constructed and it can down-regulate SIRT1 expression after transfected into PANC-1 cells.This finding could facilitate further studies on SIRT1 function and its application in tumour gene therapy.

关 键 词：胰腺肿瘤 SIRT1 RNA干扰 SHRNA 真核表达质粒 

分 类 号：R735.9[医药卫生—肿瘤]