抑制Smad3的pSUPER RNAi系统的构建和活性鉴定.pdfVIP

中南大学学报(医学版) ， Sou 西(肘耐Sci) 2007，32(6) lO42 抑制Smad3的pSUPER RNAi系统的构建和活性鉴定 张东山 ，刘伏友 ，彭佑铭 ，熊关钟 ，柴湘平 (中南大学湘雅二医院 1．肾内科；2．急诊科，长沙41001 1) [摘要】 目的：构建抑制Smad3的shRNA表达载体。方法：化学合成 2段编码短发夹RNA序列 的、靶向Smad3的寡核苷酸，各64个碱基。退火。克隆到经BgL H和Hind HI双酶切同时经牛小肠碱性 磷酸酶(alkaline phosphatase，CIP)处理后的pSUPER载体的polyllI Hl启动子的下游，重组构建 RNA干 扰(RNA induced interference，RNAi)质粒，同时设立错义寡核苷酸作为非特异性对照。并将所构建的 pSUPER Smad3转染到人类肾小管上皮细胞(human renal tubular epithelial cells，HKC)。检测其抑制效果。 结果：重组构建的pSUPER Smad3载体经双酶切电泳分析及插入基因片段序列分析，结果表明64个碱 基成功插入到预计位点，并且序列完全一致。体外活性鉴定表明其能成功地抑制Smad3的基因和蛋白 表达，却不影响Smad2的表达。结论：载体成功构建，其在体外抑制效率高。且特异性好。 [关键词】 pSUPER载体； Smad3； RNA干扰； 肾小管上皮细胞 [中图分类号] R692．9；Q784 [文献标识码] A [文章编号] 1672-7347(2007)06—1042-05 Construction and activity evaluation of pSUPER RNAi system that inhibits Smad3 ZHANG Dong—shan ’ ，LIU Fu—you ，PENG You—ming ，XIONG Guan—zhong ，CHAI Xiang—ping (1．Department ofNephrology；2．Department of Emergency，Second Xiangya Hospital， Centraf South University，Changsha 41O01 1，China) Abstract： Objective To construct the expressing vector of siRNA which can inhibit the Smad 3 activity．Methods Sixty—four bases of 2 pair oligos for hairp in RNA expression which targe— ted Smad3 gene were chemically synthesized and annealed．pSUPER vector was linearized with BgL Ⅱand Hin d 111 treated with alkaline phosphatase(CIP)．Anneled oligos were inserted into the down— stream of the treated pSUPER’S pol 111 H 1 promoter to construct RNAi plasmid(pSUPER Smad3)． Oligos with a scrambled sequence were used as a negative contro1．pSUPER Smad3 was transfected into human renal tubula~epi