Bcr/abl特异性RNA干扰真核表达载体的构建

Construction of Eukaryotic Expression Vector of RNA interference Specific for Bcr/abl

目的:构建特异性的抗慢性髓细胞白血病融合基因bcr/ablmRNA的siRNA真核细胞表达载体,探索RNA干扰(RNAi)分子靶向治疗慢性髓细胞性白血病(CML)的作用。方法:根据GenBank数据库提供的bcr/abl基因核苷酸序列,按照Tuschl设计原则,选择设计双链小干扰RNA(smallinterferingRNA,siRNA),再转化为能表达其小发卡结构RNA(SmallhairpinRNAs,shRNA)的DNA序列,并与pTER质粒定向连接,构建受控于人RNA聚合酶Ⅲ启动子H1的真核表达载体pTER/shRNA1、pTER/shRNA2,经限制性内切酶酶切和DNA测序进行鉴定。结果:构建慢性粒细胞白细胞bcr/abl融合基因siRNA真核表达载体pTER/shRNA1、pTER/shRNA2,经限制性内切酶酶切和DNA测序证实与设计完全一致。结论:抗慢性粒细胞白细胞bcr/abl融合基因siRNA真核细胞表达载体的构建成功。

Objective:To construct eukaryotic expression vector of siRNA specific for bcr/abl as a molecular target tool to cleave bcr/abl mRNA in chronic myelogenous leukemia cell, and to expore the feasibility of it. Methods:Genome sequences of bcr/abl fusion gene was retrieved from Genbank, siRNA(small interfering RNA)was designed according to the Tuschl's principle of RNAi-based medicine,and was converted into cDNA coding expression of shRNA(small hairpin RNAs)of siRNA for bcr/abl fusion gene.The cDNA was synthesized and inserted into plasmid pTER.The pTER shRNA1 and pTER shRNA2 of recombinant plasmid being eukaryotic expression vector was controlled by the H1 promoter of RNA polymerase Ⅲ,and identified by the restriction map and the sequence analysis.Results:The pTER/shRNA1 and pTER/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 bcr/abl mRNA has been successfully conctructed.