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目的:构建有效的Livin shRNA重组质粒。方法:设计、合成Livin shRNA,与pGenesil-1质粒载体链接构建重组质粒,通过酶切电泳、基因测序证实是否正确构建,通过转染高表达Livin的大肠癌HT-29细胞检测Livin mRNA下降水平,筛选出最佳的shRNA。结果:重组质粒pGenesil-shRNA经酶切电泳、基因测序证明寡核苷酸片段成功插入预计位点,且序列与我们设计合成的完全一致;重组质粒载体转染HT-29细胞后,肿瘤细胞Livin mRNA含量较转染前及对照组均有明显的下降(p<0.01),其中尤以Livin1抑制作用明显,抑制率达到66%。结论:我们正确构建了Livin ShRNA重组质粒,且制备的shRNA能有效抑制Livin基因的表达,为探讨针对Livin基因的RNAi对肿瘤的治疗奠实验基础。
Objective: To construct effective Livin shRNA recombinant plasmids. METHODS: Livin shRNA was designed and synthesized. The recombinant plasmids were constructed by ligating pGenesil-1 plasmid vector and confirmed by enzyme-digestion electrophoresis. The down-regulation of Livin mRNA was detected by transfecting Livin-overexpressing HT-29 cells. Screen out the best shRNA. Results: The recombinant plasmid pGenesil-shRNA was successfully digested by restriction endonuclease digestion and sequencing. The sequence of the gene was exactly the same as that we designed and synthesized. After transfected with HT-29 cells, the expression of Livin Compared with pre-transfection group and control group, mRNA expression decreased significantly (p <0.01), especially in Livin1 group. The inhibitory rate was 66%. Conclusion: We constructed Livin ShRNA recombinant plasmids correctly, and the prepared shRNA can effectively inhibit the expression of Livin gene, so as to lay the experimental foundation for exploring the therapeutic effect of Livin RNAi on tumor.