靶向Livin基因siRNA载体的构建和沉默效应观察

目的:构建针对人Livin基因的小干扰RNA(siRNA)表达载体,并观察该载体对食管癌细胞Livin基因表达的沉默效应。方法:依据siRNA设计原则,分析Livin mRNA序列,设计并合成2个Livin靶向的发夹状siRNA靶序列,退火后分别连接入pSUPER-neo载体,转化扩增后进行序列测定。用脂质体包裹法将重组质粒转染人食管癌细胞EC9706,采用RT-PCR检测转染细胞Livin mRNA的表达。结果:经过酶切鉴定与测序,构建的靶向Livinα和Livinβ基因的siRNA载体,结果正确;RT-PCR检测显示,转染细胞Livin mRNA的表达水平降低。结论:成功构建沉默细胞Livin基因表达的siRNA载体。

Construction of siRNA vector targeting human Livin gene and observation of its silence efficiency

Aim:To construct the siRNA expression vector targeted human Livin gene and observe its effciency.Methods:Specific short chain oligonucletide was designed according to the mRNA sequence,the double chain DNA sequenc was gained through annealing after chemosynthesis and was inserted to pSUPER-neo vector.The recombinant expression vector was transfected into human esophageal carcinoma cell line EC9706 by Liposome.The Livin mRNA in transfected cells were determined by RT-PCR.Results:Identificated by enzyme cutting and sequencing,the siRNA expression vector targeted human Livin gene was constructed successfully.Livin mRNA expression level of transfected cells were dramatically suppressed.Conclusion:The constructed siRNA expression vector can silence Livin gene expression effectively.