三位点GSK3β shRNA 真核表达质粒的构建及鉴定
Construction of Eukaryotic Vector Expressing Three shRNAs of GSK3β Gene
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摘要: 目的：构建三位点干扰GSK3β基因的高效shRNA表达质粒载体。方法：用DNA重组技术将针对人GSK3β基因不同部位所设计的3对shRNA序列克隆到高效RNAi真核表达质粒载体pshRNA-U3中，构建shRNA表达载体pshRNA-U3/GSK3β。脂质体介导转染人卵巢癌细胞株OV2008，流式细胞仪检测转染效率。运用实时定量PCR和Western blot分别检测RNA水平和蛋白水平GSK3β的干扰效果。运用流式细胞仪检测紫杉醇诱导的凋亡率。结果：经测序证实成功构建携带3个GSK3β shRNA的表达载体pshRNA-U3/GSK3β。RT-PCR和Western blotting均证实重组质粒pshRNA-U3/GSK3β转染OV2008后可明显降低细胞内GSK3β mRNA丰度及GSK3β蛋白表达。FACS显示，干扰GSK3β可抵抗紫杉醇诱导的凋亡。结论：成功构建了针对GSK3β的三位点shRNA表达载体pshRNA-U3/GSK3β，并进行了初步的功能效应验证，为进一步研究GSK3β 蛋白分子的生物学功能及应用奠定了基础。Abstract: Objective：To explore the feasibility of selective inhibiting GSK3β expression using GSK3β short hairpin RNA (shRNA) interference. Methods：Three 19bp reverse repeated motifs targeted to GSK3β gene were synthesized and cloned into eukaryotic expression plasmid pshRNA -U3 containing three U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pshRNA-U3/GSK3β and pshRNA-U3/con were transfected into OV2008 cells by lipofectamine reagent，respectively.The alteration of GSK3β expression was examined by real time-PCR and Western blot. Results：It was verified by partial nucleotide sequencing that the constructed eukaryotic vector expressing three shRNA of GSK3β was correct.OV2008 cellstransfected by pshRNA-U3/GSK3β dramatically down-regulated the expression of GSK3β mRNA and protein compared with untransfected and control cells. Conclusion：The shRNA can efficiently suppress expression of GSK3β in OV2008 cells. The results of the study lay the foundation for further studying on biological functions and potential application of GSK3β.
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