Livin基因短发卡RNA表达质粒的构建及其对胶质瘤Livin基因表达的影响

目的构建凋亡抑制基因livin基因的特异性短发卡RNA（siRNA）真核表达载体,并观察其在人脑胶质瘤细胞中对livin基因表达的抑制。方法设计有小发夹结构的2条livinβ siRNA对应的DNA序列,将其克隆入pSliencer 3.1质粒,构建重组质粒pSliencer-livinβ,对重组质粒进行酶切分析和DNA序列测定。以脂质体法将pSliencer-livinβ转染人胶质瘤细胞。采用RT-PCR和Western-blot检测Livinβ蛋白的表达,筛选最有效的一组pSliencer-livinβ质粒。结果酶切及测序证实质粒pSliencer-livinβ构建成功。转染后胶质瘤细胞livinβmRNA和蛋白表达均受到明显抑制。结论成功构建livinβ基因的特异性短发卡RNA（siRNA）真核表达载体能够显著抑制人胶质瘤细胞livinβ基因的表达。

Construction of plasmid expressive vector coding short hairpin RNA against livin gene and its effect on the expression of livin gene in glioma cells

Objective To construct the specific short hairpin RNA(siRNA) eukaryotic plasmid expressive vector targeting human livin gene and to observe its inhibitory effect livin gene in the GL15 glioma cells.Methods Two livin|? siRNA template DNA sequences were designed and synthesized.The annealed siRNA template was cloned into pSliencer 3.1 plasmid.The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing.The recombinant plasmid was transfected by liposome mediated transfection into GL15 glioma cells.The effect on the livin gene expression in GL15 glioma cell was detected by RT-PCR and Western blotting.The most effective pSliencer-livin |? plasmid was selected.Results It was confirmed by restrictive enzyme digestion and sequence analysis that recombinant plasmid was cloned successfully.Expressions of livin |? mRNA and protein in glioma cells were suppressed after transfection.Conclusion The siRNA eukaryotic expression vector targeting human livin gene could be constructed successfully and can remarkably inhibit the expression of livin |? gene in human glioma cells.