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本研究构建并鉴定靶向mdr-1基因的短发夹RNA(shRNA)真核表达载体。根据Genbank数据库提供的mRNA序列,选择设计3个能转录短发夹RNA(shRNA)的DNA序列,体外分别合成3对互补并编码mRNA基因的特异性shRNA序列的寡核苷酸,克隆到经BamHI和HindⅢ双酶切线性化的pGCsilencer人U6启动子质粒载体中;为了确定利用酶切和测序鉴定所构建的重组载体是否正确,采用脂质体转染的方法将构建好的3个重组载体(pGY1-1,pGY1-2,pGY1-3)导入慢性髓系白血病耐药细胞株K562/A02,采用实时PCR方法检测转染细胞的mdr-1基因表达,Western-blot检测P-gp表达量的变化。结果表明:应用PCR电泳图谱和测序证实了克隆RNAi打靶序列完全正确,而且该载体又同时表达GFP标记基因,用于测定转染效率。分别转染pGY1-1,pGY1-2,pGY1-3后K562/A02细胞的mdr-1基因表达和P-gp表达水平下降,证明shRNA可以特异性沉默mdr-1基因。结论:靶向mdr-1基因shRNA真核表达载体构建成功,为进一步研究mdr-1基因在K562/A02细胞中的作用奠定了实验基础。
In this study, a short hairpin RNA (shRNA) eukaryotic expression vector targeting mdr-1 gene was constructed and identified. According to the mRNA sequence provided by GenBank, three DNA sequences capable of transcribing short hairpin RNAs (shRNAs) were designed and synthesized. Three pairs of oligonucleotides complementary to specific shRNA sequences encoding mRNA genes were synthesized in vitro and cloned into BamHI And Hind Ⅲ double digestion linearization pGCsilencer human U6 promoter plasmid vector; in order to confirm the use of restriction enzyme digestion and sequencing to identify the constructed recombinant vector is correct, liposome transfection method will be constructed three recombinant vectors ( pGY1-1, pGY1-2 and pGY1-3) were transfected into K562 / A02 cells, and mdr-1 gene expression was detected by real-time PCR. The expression of P-gp Variety. The results showed that cloned RNAi targeting sequence was confirmed by PCR electrophoresis and sequencing. The vector also expressed GFP marker gene simultaneously, which was used to determine the transfection efficiency. After transfected with pGY1-1, pGY1-2 and pGY1-3 respectively, mdr-1 gene expression and P-gp expression decreased in K562 / A02 cells, which demonstrated that shRNA could specifically silence mdr-1 gene. CONCLUSION: The eukaryotic expression vector targeting shRNA of mdr-1 gene was successfully constructed, which laid the experimental foundation for further study of the role of mdr-1 in K562 / A02 cells.