USP22基因启动子的克隆与鉴定
发布时间：2019-06-10 02:21
【摘要】：目的：对泛素水解酶22（Ubiquitin-specific processing enzyme22，USP22）基因转录起始位点上游启动子区域进行克隆与鉴定，界定出USP22基因核心启动子的范围，为USP22基因表达调控的深入研究奠定基础。 方法：运用cDNA5'末端快速扩增（5'RACE）方法对USP22基因转录起始位点的确定。以人基因组为模板，利用巢氏PCR方法对USP22基因5'端侧翼区包括转录起始点在内进行扩增，PCR产物两端分别引入KpnI和BglII酶切位点。凝胶回收扩增产物，TA克隆，DNA测序鉴定扩增片段正确无误。将PCR扩增产物定向克隆入荧光素酶表达载体PGL-3Basic，，构建含USP22启动子序列和荧光素酶报告基因的重组质粒。脂质体介导以上重组质粒与内参质粒PRL-TK共同转染HepG2细胞和HeLa细胞，运用荧光素酶报告基因分析法，检测不同片段启动子转录活性，同时设立阴性对照组(PGL-3Basic)。对所得数据进行统计分析，鉴定出USP22核心启动子区域。 结果：鉴定USP22只有一个转录起始点，位于翻译起始点ATG上游-176bp。克隆出8个USP22启动子片段，酶切及测序结果证实USP22启动子系列递减片段插入到荧光素酶报告基因载体PGL-3Basic中，构建USP22promoter/PGL-3Basic真核表达载体。重组质粒瞬时转染入HepG2细胞和HeLa细胞后，荧光素酶检测结果表明USP22promoter在肿瘤细胞中具有很强的转录活性，调控USP22基因表达的核心启动子片段推测是-210～-7区域，且在-595～-326之间可能有正性调控序列，在-866～-595之间可能有抑制性DNA元件。 结论：确定USP22基因转录起始位点（Transcription starting sites，TSS），克隆和鉴定USP22基因的核心启动子，为进一步研究对USP22转录调控起关键作用的DNA元件的深入研究奠定基础。
[Abstract]:Aim: to clone and identify the upstream promoter region of ubiquitin hydrolase 22 (Ubiquitin-specific processing enzyme22,USP22) gene transcriptional initiation site, and to define the range of USP22 gene core promoter, so as to lay a foundation for further study of USP22 gene expression regulation. Methods: the transcriptional initiation site of USP22 gene was determined by cDNA5' terminal rapid amplification (5'RACE). Using the human genome as template, the 5 'flanking region of USP22 gene, including the transcriptional starting point, was amplified by nest PCR. KpnI and BglII restriction sites were introduced into both ends of PCR products, respectively. The amplified products were recovered by gel, cloned by TA and identified by DNA sequencing. The PCR products were cloned into luciferase expression vector PGL-3Basic, to construct a recombinant plasmid containing USP22 promoter sequence and luciferase reporter gene. HepG2 cells and HeLa cells were co-transfected with the recombinant plasmid PRL-TK mediated by liposome. luciferase reporter gene analysis was used to detect the transcriptional activity of different promoter fragments, and a negative control group (PGL-3Basic) was set up at the same time. The core promoter region of USP22 was identified by statistical analysis of the data. Results: there was only one transcriptional starting point in USP22, which was located in the upstream of ATG. Eight USP22 promoter fragments were cloned. The results of restriction enzyme digestion and sequencing confirmed that the USP22 promoter series decreasing fragments were inserted into luciferase reporter gene vector PGL-3Basic to construct USP22promoter/PGL-3Basic eukaryotic expression vector. After transient transfer of recombinant plasmid into HepG2 cells and HeLa cells, luciferase assay showed that USP22promoter had strong transcriptional activity in tumor cells, and the core promoter fragment regulating the expression of USP22 gene was speculated to be-210 鈮
本文编号：2496091
[Abstract]:Aim: to clone and identify the upstream promoter region of ubiquitin hydrolase 22 (Ubiquitin-specific processing enzyme22,USP22) gene transcriptional initiation site, and to define the range of USP22 gene core promoter, so as to lay a foundation for further study of USP22 gene expression regulation. Methods: the transcriptional initiation site of USP22 gene was determined by cDNA5' terminal rapid amplification (5'RACE). Using the human genome as template, the 5 'flanking region of USP22 gene, including the transcriptional starting point, was amplified by nest PCR. KpnI and BglII restriction sites were introduced into both ends of PCR products, respectively. The amplified products were recovered by gel, cloned by TA and identified by DNA sequencing. The PCR products were cloned into luciferase expression vector PGL-3Basic, to construct a recombinant plasmid containing USP22 promoter sequence and luciferase reporter gene. HepG2 cells and HeLa cells were co-transfected with the recombinant plasmid PRL-TK mediated by liposome. luciferase reporter gene analysis was used to detect the transcriptional activity of different promoter fragments, and a negative control group (PGL-3Basic) was set up at the same time. The core promoter region of USP22 was identified by statistical analysis of the data. Results: there was only one transcriptional starting point in USP22, which was located in the upstream of ATG. Eight USP22 promoter fragments were cloned. The results of restriction enzyme digestion and sequencing confirmed that the USP22 promoter series decreasing fragments were inserted into luciferase reporter gene vector PGL-3Basic to construct USP22promoter/PGL-3Basic eukaryotic expression vector. After transient transfer of recombinant plasmid into HepG2 cells and HeLa cells, luciferase assay showed that USP22promoter had strong transcriptional activity in tumor cells, and the core promoter fragment regulating the expression of USP22 gene was speculated to be-210 鈮
本文编号：2496091
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