Survivin特异性siRNA真核表达载体的构建

The Construction of Eukaryotic Expression Vector of siRNA Specific for Survivin Gene

目的构建Survivin特异性siRNA真核表达载体,为以Survivin基因为靶点、小干扰RNA(small interfering RNA,siRNA)为手段的白血病和其它恶性肿瘤基因治疗的基础和临床应用提供良好的技术手段。方法根据GeneBank数据库提供的Survivin基因所有变异体的共同核苷酸序列,按照Tuschl设计原则,选择设计双链siRNA,再转化为能表达其小发卡结构RNA(small hairpinRNA,shRNA)的DNA序列,并与载体pSINsi-hU6定向连接,构建受控于启动子U6的真核表达载体pSIN/shRNA1、pSIN/shR-NA2,经限制性内切酶酶切和DNA测序进行鉴定。结果构建Survivin特异性siRNA真核表达载体pSIN/shRNA1、pSIN/shR-NA2,经限制性内切酶酶切和DNA测序证实与设计完全一致。结论Survivin特异性siRNA真核表达载体构建成功。

Objective To construct eukaryotic expression vector of siRNA specific for Survivin gene as a molecular target tool to clinic of leukemia and other malignant tumor. Methods Genome sequences of Survivin gene was retrieved from gene bank, siRNA （ small interfering RNA） was designed according to the Tuschl＇s principle of RNAi-based medicine, and was converted into eDNA coding expression of shRNA （small hairpin RNA） of siRNA for Survivin gene. The eDNA was synthesized and inserted into plasmid pSINsi-hU6. The pSIN/shRNA1 and pSIN/shRNA2 of recombinant plasmid being eukaryotic expression vector was controlled by the U6 promoter, and identified by the restriction map and the sequence analysis. ResuLts The pSIN/ shRNA1 and pSIN/ shRNA2 of recombinant plasmid identificated by the restriction map and the sequence analysis completely coincided with the designs. ConcLusions The siRNA eukaryotic expression vector against Survivin gene has been successfully conctructed.