bcr/abl特异性siRNA真核表达载体的构建及鉴定

Construction of eukaryotic expression vector of siRNA specific to bcr/abl fusion gene

目的:构建bcr/abl的特异性siRNA真核细胞表达载体,并初步探索对K562细胞bcr/abl mRNA和P210蛋白的影响。方法:根据GenBank数据库提供的bcr/abl基因核苷酸序列,按照Tusch l设计原则,选择设计双链小干扰RNA(siRNA),再转化为能表达其小发卡结构RNA(shRNA)的DNA序列,并与pTER质粒定向连接,构建受控于人RNA聚合酶启动子H1的真核表达载体pTER117,经限制性内切酶酶切和DNA测序进行鉴定;在脂质体的介导下转染K562细胞,用RT-PCR分析bcr/abl mRNA的表达,细胞化学染色检测P210蛋白的表达。结果:构建bcr/abl融合基因siRNA真核表达载体pTER117经限制性内切酶酶切和DNA测序证实与设计完全一致,转染K562细胞24 h后,pTER117使bcr/abl mRNA的相对水平下降50%,使P210蛋白下降47%。结论:bcr/abl融合基因siRNA真核细胞表达载体构建成功,并有效干扰K562细胞bcr/abl的表达。

Objective ：To construct eukaryotie expression vector of siRNA specific to ber/abl and to initially investigate the effect of recombinant plasmid on bcr/abl and P210 protein expression in K562 cells. Methods：siRNA （ small interfering RNA）was designed according to the tuschl＇s principle of RNA i-based medicine,and was converted into cDNA coding expression of shRNA （small hairp in RNA） of siRNA for ber/a blfusion gene. The cDNA was synthesized and inserted into plasmid pTER. The pTER117 of recombinant plasmid being eukaryotie expression vector was controlled by the H1 promoter of RNA polymerase,identified by the restriction map and the sequence analysis, and transfeeted into K562 cells by lipofeetamine. Expression of bcr/abl mRNA was assayed by RT-PCR,expression of P210 protein w as detected by immuno histochemistry. Results：The pTER117 of recombinant plasmid identified by the restriction map and the sequence analysis completely coincided with the designs. 24 hours after transfection in K562 cells,the recombinant plasmid could down regulate the expression of the bcr/abl mRNA and bcr/abl protein （P210） in K562 cells. Conclusion ：The siRNA eukaryotic expression vector against bcr/abl mRNA has been successfully conctructed,and it effectively inhibits the expression of bcr/abl in K562 cells.