Livin shRNA质粒载体的构建及评价

Construction and Evaluation of Recombinant Plasmid of Livin shRNA

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 an effective recombinant plasmid of Livin shRNA. Methods：Livin shRNA was designed and synthetized,then linked with pGenesil-1 plasmid vector to construct a recombinant plasmid. At last we verified that the correct plasmid vector has been constructed by enzyme-cutting electro-phpresis and gene sequencing.The plasmid was transfected into HT-29 cell which express highly of Livin and the mRNA was detected to select the optmal shRNA. Results：Oligonucleotides were correctly inserted into the recombinant plasmid pGenesil-shRNA by enzyme-cutting electro-phoresis and gene sequencing and the sequence was completely the same as designed.After the recombinant plasmid vector was transfected into HT-29 cell,livin mRNA level of cancer cell was remarkably decreased than before transfection and control group（p〈0.01） .It verified that the shRNA prepared can inhibit the express of Livin efficiently and the role of livin1 is more obvious,the inhibition rate of which was 66 percent. Conclusion：We constructed correctly the recombinant plasmid of Livin shRNA,which can inhibit the express of Livin efficiently .It also established the experiement foundation that explore the tumorous gene therapy of RNAi to Livin.