稳定表达人α-分泌酶adam10基因启动子荧光素酶报告基因细胞系的构建研究
发布时间：2018-08-09 20:05
【摘要】：目的构建携带adam10基因启动子的荧光素酶报告载体,筛选稳定表达细胞系并分析其活性。方法提取人神经母细胞瘤细胞(SH-SY5Y细胞)基因组DNA,以其为模板,PCR扩增adam10基因启动子并克隆至荧光素酶报告载体pGL4.17中,构建adam10基因启动子荧光素酶报告载体pGL4.17-adam10,将其转染SH-SY5Y细胞(无启动子的pGL4.17载体作阴性对照,带有CMV启动子的pGL4.51载体作阳性对照),经G418进行稳定表达株的筛选,用1μmol/L维甲酸处理细胞4d后检测其荧光活性。结果成功扩增到438bp的adam10基因启动子,pGL4.17-adam10经PCR和双酶切鉴定均正确。SH-SY5Y细胞被该载体转染后经G418筛选得到稳定表达adam10基因启动子的细胞株,经检测具有较强的转录活性;1μmol/L维甲酸能诱导adam10基因启动子高效表达。结论成功构建了人adam10基因启动子荧光素酶报告载体,adam10基因启动子在SH-SY5Y细胞中能稳定表达,为深入研究adam10基因的表达调控、多态性分析及其高通量药物筛选提供基础。
[Abstract]:Objective to construct a luciferase report vector carrying the promoter of adam10 gene and to screen stable expression cell lines and analyze their activity. Methods the genomic DNA of human neuroblastoma cells (SH-SY5Y cells) was extracted. The promoter of adam10 gene was amplified by PCR and cloned into luciferase report vector pGL4.17. Adam10 gene promoter luciferase report vector pGL4.17-adam10 was constructed and transfected into SH-SY5Y cells (pGL4.17 vector without promoter as negative control and pGL4.51 vector with CMV promoter as positive control). The fluorescence activity of the cells treated with 1 渭 mol/L retinoic acid for 4 days was determined. Results the adam10 gene promoter pGL4.17-adam10 of 438bp was successfully amplified and identified by PCR and double enzyme digestion. After transfection of SH-SY5Y cells by the vector, the cell lines expressing adam10 gene promoter stably were screened by G418. 1 渭 mol/L retinoic acid with a strong transcriptional activity could induce high expression of adam10 gene promoter. Conclusion the luciferase reporter vector of human adam10 gene luciferase can be stably expressed in SH-SY5Y cells, which provides a basis for further study on the regulation of adam10 gene expression, polymorphism analysis and high throughput drug screening.
【作者单位】： 重庆医科大学附属第一医院神经内科;
【分类号】：R749.16
[Abstract]:Objective to construct a luciferase report vector carrying the promoter of adam10 gene and to screen stable expression cell lines and analyze their activity. Methods the genomic DNA of human neuroblastoma cells (SH-SY5Y cells) was extracted. The promoter of adam10 gene was amplified by PCR and cloned into luciferase report vector pGL4.17. Adam10 gene promoter luciferase report vector pGL4.17-adam10 was constructed and transfected into SH-SY5Y cells (pGL4.17 vector without promoter as negative control and pGL4.51 vector with CMV promoter as positive control). The fluorescence activity of the cells treated with 1 渭 mol/L retinoic acid for 4 days was determined. Results the adam10 gene promoter pGL4.17-adam10 of 438bp was successfully amplified and identified by PCR and double enzyme digestion. After transfection of SH-SY5Y cells by the vector, the cell lines expressing adam10 gene promoter stably were screened by G418. 1 渭 mol/L retinoic acid with a strong transcriptional activity could induce high expression of adam10 gene promoter. Conclusion the luciferase reporter vector of human adam10 gene luciferase can be stably expressed in SH-SY5Y cells, which provides a basis for further study on the regulation of adam10 gene expression, polymorphism analysis and high throughput drug screening.
【作者单位】： 重庆医科大学附属第一医院神经内科;
【分类号】：R749.16
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