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中国细胞生物学学报 Chinese Journal of Cell Biology 2013, 35(2): 175–179,187
SV40增强子对奶牛β-酪蛋白启动子活性的影响
钟宇1 邵正2 江黎明3*
(1广东医学院分析中心, 湛江 524023; 2广东医学院寄生虫学教研室, 湛江 524023; 3广东医学院生物化学
与分子生物学研究所, 湛江 524023)
摘要该文采用重叠PCR方法在奶牛β-酪蛋白启动子(op0)中插入SV40增强子构建重组启
动子op0-SV40enh, 并分析其活性。首先PCR扩增op0 5′- Kb片段、op0 3′-端1 Kb片段和SV40
增强子序列, 重叠PCR拼接三种片段得到插入SV40增强子的op0-SV40enh启动子并测序鉴定后, 酶
切连接将其插入pGL3-Basic中的指定克隆位点, 构建重组载体pGL3-op0-SV40enh。将重组载体
pGL3-op0和pGL3-op0-SV40enh分别瞬时转染乳腺癌MCF-7细胞, 采用双荧光素酶报告基因检测系
统检测启动子op0和op0-SV40enh的相对活性。结果显示, Kb的片段, 测
序结果与预期结果一致, 表明成功构建了重组载体pGL3-op0-SV40enh; op0-SV40enh启动子的活性
远高于op0启动子的活性, 表明奶牛β-酪蛋白启动子中插入SV40增强子序列可显著提高其引导荧
光素酶报告基因表达的活性。
关键词 SV40增强子; 奶牛op0; 双荧光素酶报告基因检测系统
Effect of SV40 Enhancer on Cow β-casein Promoter Activity
Zhong Yu1, Shao Zheng2, Jiang Liming3*
_
x±s (1Analysis Center of Guangdong Medical College, Zhanjiang 524023, China; 2Department of Parasitology, Guangdong
Medical College, Zhanjiang 524023, China; 3Institute of Biochemistry and Molecular Biology, Guangdong Medical College,
Zhanjiang 524023, China)
Abstract By overlapping PCR method SV40 enhancer was inserted into the op0 promoter to construct re-
combinant promoter op0-SV40enh, and its activity was analyzed. Firstly, PCR amplified 5′- Kb fragment of cow
β-casein promoter, 3′-1 Kb fragment of cow β-casein promoter and SV40 enhancer sequence. Secondly