热激启动子驱动的真核表达质粒pHSP-shPLK1-GFP的构建与鉴定
发布时间：2018-11-22 09:31
【摘要】：目的构建热激启动子(p HSP)驱动的靶向敲低Polo样蛋白激酶1(PLK1)的真核表达质粒,并观察其对骨肉瘤U2OS细胞增殖的影响。方法首先合成热休克蛋白HSP70的启动子,克隆至带有绿色荧光蛋白(GFP)标签的真核表达质粒p EGFP-C1中,利用p HSP替换原有的CMV启动子,构建真核表达载体p HSP-GFP;其次,利用RNA干扰技术合成以plk1基因为靶向目标的sh RNA,将其定向克隆至真核表达载体p HSP-GFP中形成重组质粒p HSP-sh PLK1-GFP;另外设计一组阴性对照质粒p HSP-NC-GFP,最后,利用脂质体Lipo2000转染的方法将质粒转染至U2OS细胞,非热激组细胞正常培养,热激组及阴性对照组细胞42℃加热2 h,通过细胞免疫荧光染色实验检测GFP的表达,从而确定p HSP的驱动能力,采用Real-time PCR法检测细胞内plk1的m RNA表达水平,Western blot法检测PLK1蛋白表达水平,MTT法检测重组质粒对U2OS细胞增殖的影响。结果成功构建了p HSP驱动的真核表达质粒p HSP-sh PLK1-GFP;细胞免疫荧光染色实验结果显示p HSP可正常驱动gfp基因的表达,且GFP蛋白定位在胞质中;Real-time PCR结果显示热激组细胞plk1基因表达量明显降低,与非热激组及阴性对照组相比差异有统计学意义(P0.05);Western blot结果显示热激组细胞PLK1蛋白表达量比非热激组及阴性对照组低(P0.05);MTT结果显示热激组细胞的增殖活性被明显抑制,与非热激组及阴性对照组相比差异有统计学意义(P0.05)。结论 p HSP驱动的真核表达质粒p HSP-sh PLK1-GFP能在U2OS细胞中表达,在42℃加热条件下,该重组质粒呈现更强的下调plk1基因表达和抑制增殖作用。
[Abstract]:Objective to construct the eukaryotic expression plasmid of Polo like protein kinase 1 (PLK1) driven by heat shock promoter (p HSP) and to observe its effect on the proliferation of U2OS cells in osteosarcoma. Methods the promoter of heat shock protein HSP70 was synthesized and cloned into the eukaryotic expression plasmid p EGFP-C1 with green fluorescent protein (GFP) tag. The original CMV promoter was replaced by p HSP, and the eukaryotic expression vector p HSP-GFP; was constructed. Secondly, sh RNA, targeting plk1 was synthesized by RNA interference technique and cloned into eukaryotic expression vector p HSP-GFP to form recombinant plasmid p HSP-sh PLK1-GFP;. In addition, a group of negative control plasmids p HSP-NC-GFP, were designed. Finally, the plasmids were transfected into U2OS cells by liposome Lipo2000 transfection. The cells in non-heat shock group were cultured normally, and the cells in heat shock group and negative control group were heated at 42 鈩，
本文编号：2348876
[Abstract]:Objective to construct the eukaryotic expression plasmid of Polo like protein kinase 1 (PLK1) driven by heat shock promoter (p HSP) and to observe its effect on the proliferation of U2OS cells in osteosarcoma. Methods the promoter of heat shock protein HSP70 was synthesized and cloned into the eukaryotic expression plasmid p EGFP-C1 with green fluorescent protein (GFP) tag. The original CMV promoter was replaced by p HSP, and the eukaryotic expression vector p HSP-GFP; was constructed. Secondly, sh RNA, targeting plk1 was synthesized by RNA interference technique and cloned into eukaryotic expression vector p HSP-GFP to form recombinant plasmid p HSP-sh PLK1-GFP;. In addition, a group of negative control plasmids p HSP-NC-GFP, were designed. Finally, the plasmids were transfected into U2OS cells by liposome Lipo2000 transfection. The cells in non-heat shock group were cultured normally, and the cells in heat shock group and negative control group were heated at 42 鈩，
本文编号：2348876
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