穩(wěn)定表達人α-分泌酶adam10基因啟動子熒光素酶報告基因細胞系的構建研究
發(fā)布時間：2018-08-09 20:05
【摘要】：目的構建攜帶adam10基因啟動子的熒光素酶報告載體,篩選穩(wěn)定表達細胞系并分析其活性。方法提取人神經母細胞瘤細胞(SH-SY5Y細胞)基因組DNA,以其為模板,PCR擴增adam10基因啟動子并克隆至熒光素酶報告載體pGL4.17中,構建adam10基因啟動子熒光素酶報告載體pGL4.17-adam10,將其轉染SH-SY5Y細胞(無啟動子的pGL4.17載體作陰性對照,帶有CMV啟動子的pGL4.51載體作陽性對照),經G418進行穩(wěn)定表達株的篩選,用1μmol/L維甲酸處理細胞4d后檢測其熒光活性。結果成功擴增到438bp的adam10基因啟動子,pGL4.17-adam10經PCR和雙酶切鑒定均正確。SH-SY5Y細胞被該載體轉染后經G418篩選得到穩(wěn)定表達adam10基因啟動子的細胞株,經檢測具有較強的轉錄活性;1μmol/L維甲酸能誘導adam10基因啟動子高效表達。結論成功構建了人adam10基因啟動子熒光素酶報告載體,adam10基因啟動子在SH-SY5Y細胞中能穩(wěn)定表達,為深入研究adam10基因的表達調控、多態(tài)性分析及其高通量藥物篩選提供基礎。
[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.
【作者單位】： 重慶醫(yī)科大學附屬第一醫(yī)院神經內科;
【分類號】：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.
【作者單位】： 重慶醫(yī)科大學附屬第一醫(yī)院神經內科;
【分類號】：R749.16
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