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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/ablmRNA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聚合酶启动子H1的真核表达载体pTER117、pTER363,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/ablmRNA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、pTER363,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24h后,pTER117、pTER363分别使bcr/ablmRNA的相对水平下降52%、43%,使P210蛋白分别下降47%、40%。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 specific expression vector of bcr / abl siRNA eukaryotic cells and to explore the effects on bcr / abl mRNA and P210 protein in K562 cells. Methods According to the nucleotide sequence of bcr / abl gene provided by GenBank database, double-stranded small interfering RNA (siRNA) was designed according to Tuschl design principle and then transformed into DNA that can express small hairpin RNAs (shRNAs) Sequence and ligated with pTER plasmid to construct eukaryotic expression vectors pTER117 and pTER363 which are controlled by human RNA polymerase promoter H1 and confirmed by restriction endonuclease digestion and DNA sequencing. K562 cells were transfected with Bcr / ablmRNA by RT-PCR. The expression of P210 protein was detected by cytochemical staining. Results The eukaryotic expression vectors pTER117 and pTER363 of bcr / abl fusion gene siRNA were constructed. The restriction endonuclease digestion and DNA sequencing confirmed that the expression of bcr / abl mRNA was consistent with the design. After transfection with K562 cells for 24 h, pTER117 and pTER363 increased the relative The levels decreased by 52% and 43%, respectively, which decreased the P210 protein by 47% and 40% respectively. Conclusion The eukaryotic expression vector of bcr / abl fusion gene siRNA was successfully constructed and effectively inhibited the expression of bcr / abl in K562 cells.