穩(wěn)定表達(dá)人α-分泌酶adam10基因啟動(dòng)子熒光素酶報(bào)告基因細(xì)胞系的構(gòu)建研究
發(fā)布時(shí)間：2018-08-09 20:05
【摘要】：目的構(gòu)建攜帶adam10基因啟動(dòng)子的熒光素酶報(bào)告載體,篩選穩(wěn)定表達(dá)細(xì)胞系并分析其活性。方法提取人神經(jīng)母細(xì)胞瘤細(xì)胞(SH-SY5Y細(xì)胞)基因組DNA,以其為模板,PCR擴(kuò)增adam10基因啟動(dòng)子并克隆至熒光素酶報(bào)告載體pGL4.17中,構(gòu)建adam10基因啟動(dòng)子熒光素酶報(bào)告載體pGL4.17-adam10,將其轉(zhuǎn)染SH-SY5Y細(xì)胞(無(wú)啟動(dòng)子的pGL4.17載體作陰性對(duì)照,帶有CMV啟動(dòng)子的pGL4.51載體作陽(yáng)性對(duì)照),經(jīng)G418進(jìn)行穩(wěn)定表達(dá)株的篩選,用1μmol/L維甲酸處理細(xì)胞4d后檢測(cè)其熒光活性。結(jié)果成功擴(kuò)增到438bp的adam10基因啟動(dòng)子,pGL4.17-adam10經(jīng)PCR和雙酶切鑒定均正確。SH-SY5Y細(xì)胞被該載體轉(zhuǎn)染后經(jīng)G418篩選得到穩(wěn)定表達(dá)adam10基因啟動(dòng)子的細(xì)胞株,經(jīng)檢測(cè)具有較強(qiáng)的轉(zhuǎn)錄活性;1μmol/L維甲酸能誘導(dǎo)adam10基因啟動(dòng)子高效表達(dá)。結(jié)論成功構(gòu)建了人adam10基因啟動(dòng)子熒光素酶報(bào)告載體,adam10基因啟動(dòng)子在SH-SY5Y細(xì)胞中能穩(wěn)定表達(dá),為深入研究adam10基因的表達(dá)調(diào)控、多態(tài)性分析及其高通量藥物篩選提供基礎(chǔ)。
[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(xué)附屬第一醫(yī)院神經(jīng)內(nèi)科;
【分類號(hào)】：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(xué)附屬第一醫(yī)院神經(jīng)內(nèi)科;
【分類號(hào)】：R749.16
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