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  

Construction of SH-SY5Y cell line stably expressing humanα-secretase adam10 gene promoter luciferase reporter gene

作　　者：唐颖 胡小童 朱炳林[1] 韩宇[1] 

机构地区：[1]重庆医科大学附属第一医院神经内科,400016

出　　处：《重庆医学》2017年第28期3947-3949,3952,共4页Chongqing medicine

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。Objective struet the luciferase report vector carrying a disintegrin and metalloprotease 10（adam10） gene promoter, to screen its stable expression cell line and to analyze its activity. Methods The genome DNA of human neuroblastoma SH-SY5Y cells was extracted as the template. The adain10 gene promoter was amplified by PCR and was cloned into luciferase reporter vector pGL4.17. The adam 10 gene promoter luciferase reporter vector pGL4.17-adam10 was constructed and transfected in to SH-SY5Y cells（pGL4. 17 vector without promotoer as the negative control and pGL4. 17 vector with CMV promoter as the positive control）. Then the stable expression celt line was screened by G418 and its fluorescence activity was detect after treating with l μmol/L retinoic acid（RA） for 4 d.Results About 438 bp adam10 gene promoter was successfully amplified by PCR. The pGL4. 17-adam10 vector was correct by PCR and double enzyme digestion identification. The cell line stably expressing adam10 gene promoter was obtained after transfeeting SH-SYSY ceils by this vector and screening by G418, which had stronger transcriptional activity by detection;1 μmol/L RA could induce high efficiency expression of adam10 gene promoter. Conclusion Human adam10 gene promoter luciferase vector is successfully constructed..adam10 gene promoter can be stably expressed in SH-SY5Y cells, which provides a basis for deeply studying adam10 gene expression regulation, polymorphism analysis and high-throughput drug sexeening.

关 键 词：去整合素和金属蛋白酶10 启动子 荧光素酶报告载体 高通量药物筛选 

分 类 号：R749.16[医药卫生—神经病学与精神病学]