USP22基因啟動子的克隆與鑒定
發(fā)布時間：2019-06-10 02:21
【摘要】：目的：對泛素水解酶22（Ubiquitin-specific processing enzyme22，USP22）基因轉(zhuǎn)錄起始位點上游啟動子區(qū)域進(jìn)行克隆與鑒定，界定出USP22基因核心啟動子的范圍，為USP22基因表達(dá)調(diào)控的深入研究奠定基礎(chǔ)。 方法：運用cDNA5'末端快速擴(kuò)增（5'RACE）方法對USP22基因轉(zhuǎn)錄起始位點的確定。以人基因組為模板，利用巢氏PCR方法對USP22基因5'端側(cè)翼區(qū)包括轉(zhuǎn)錄起始點在內(nèi)進(jìn)行擴(kuò)增，PCR產(chǎn)物兩端分別引入KpnI和BglII酶切位點。凝膠回收擴(kuò)增產(chǎn)物，TA克隆，DNA測序鑒定擴(kuò)增片段正確無誤。將PCR擴(kuò)增產(chǎn)物定向克隆入熒光素酶表達(dá)載體PGL-3Basic，，構(gòu)建含USP22啟動子序列和熒光素酶報告基因的重組質(zhì)粒。脂質(zhì)體介導(dǎo)以上重組質(zhì)粒與內(nèi)參質(zhì)粒PRL-TK共同轉(zhuǎn)染HepG2細(xì)胞和HeLa細(xì)胞，運用熒光素酶報告基因分析法，檢測不同片段啟動子轉(zhuǎn)錄活性，同時設(shè)立陰性對照組(PGL-3Basic)。對所得數(shù)據(jù)進(jìn)行統(tǒng)計分析，鑒定出USP22核心啟動子區(qū)域。 結(jié)果：鑒定USP22只有一個轉(zhuǎn)錄起始點，位于翻譯起始點ATG上游-176bp�？寺〕�8個USP22啟動子片段，酶切及測序結(jié)果證實USP22啟動子系列遞減片段插入到熒光素酶報告基因載體PGL-3Basic中，構(gòu)建USP22promoter/PGL-3Basic真核表達(dá)載體。重組質(zhì)粒瞬時轉(zhuǎn)染入HepG2細(xì)胞和HeLa細(xì)胞后，熒光素酶檢測結(jié)果表明USP22promoter在腫瘤細(xì)胞中具有很強(qiáng)的轉(zhuǎn)錄活性，調(diào)控USP22基因表達(dá)的核心啟動子片段推測是-210～-7區(qū)域，且在-595～-326之間可能有正性調(diào)控序列，在-866～-595之間可能有抑制性DNA元件。 結(jié)論：確定USP22基因轉(zhuǎn)錄起始位點（Transcription starting sites，TSS），克隆和鑒定USP22基因的核心啟動子，為進(jìn)一步研究對USP22轉(zhuǎn)錄調(diào)控起關(guān)鍵作用的DNA元件的深入研究奠定基礎(chǔ)。
[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
本文編號：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
本文編號：2496091
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