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目的构建Survivin的短发夹RNA表达质粒并通过由其介导的RNA干扰来下调神经母细胞瘤细胞株SH-SY5Y细胞中Survivin的表达。方法针对人Survivin的mRNA序列设计合成两对寡核苷酸序列,退火后将其连入pBSHH1,对重组质粒pBSHH1-S1和pBSHH1-S2进行酶切和测序鉴定,然后将质粒转染SH-SY5Y细胞,运用RT-PCR和Western印迹检测Survivin基因的表达。结果酶切和测序证实目的寡核苷酸片段已被克隆到pBSHH1,pBSHH1-S1和pBSHH1-S2转染SH-SY5Y细胞后,Survivin基因的mRNA以及蛋白水平的表达量均受到明显抑制。结论成功构建了人Survivin基因的短发夹RNA表达质粒,并在SH-SY5Y细胞中验证了它们对Survivin基因的表达抑制作用,为神经母细胞瘤等肿瘤的基因治疗研究奠定了基础。
Objective To construct Survivin short hairpin RNA expression plasmid and to downregulate Survivin expression in neuroblastoma SH-SY5Y cells by RNAi mediated by it. Methods Two pairs of oligonucleotide sequences were designed and synthesized according to the mRNA sequence of human Survivin. After annealing, they were ligated into pBSHH1, and the recombinant plasmids pBSHH1-S1 and pBSHH1-S2 were identified by restriction enzyme digestion and sequencing. Then the plasmids were transfected into SH-SY5Y Cells, Survivin gene expression was detected by RT-PCR and Western blotting. Results Enzyme digestion and sequencing confirmed that the target oligonucleotide fragment was cloned into pBSHH1. The expression of Survivin mRNA and protein was significantly inhibited after transfection with pBSHH1-S1 and pBSHH1-S2 into SH-SY5Y cells. Conclusion The short hairpin RNA expression plasmid of human Survivin gene was successfully constructed and its inhibitory effect on the expression of Survivin gene was verified in SH-SY5Y cells, which laid the foundation for the gene therapy of tumors such as neuroblastoma.