【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′-端2.1 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,重叠PCR拼接出长度为3.4 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。更多还原

【Abstract】 By overlapping PCR method SV40 enhancer was inserted into the op0 promoter to construct recombinant promoter op0-SV40enh,and its activity was analyzed.Firstly,PCR amplified 5′-2.1 Kb fragment of cow β-casein promoter,3′-1 Kb fragment of cow β-casein promoter and SV40 enhancer sequence.Secondly,three fragments were connected by overlap PCR and inserted into multiply clone sites of pGL3-Basic vector and a recombinant vector named pGL3-op0-SV40enh was constructed.Finally,the activity of op0 promoter and op0-SV40enh promoter were detected by Dual-Luciferase Reporter Assay System.Overlapping PCR spliced out of the length of 3.4 Kb fragments,and the sequencing results consistent with the expected results.These indicated that the recombinant pGL3-op0-SV40enh vector be constructed successfully.The results of Dual-Luciferase Reporter Assay indicated that Op0-SV40enh promoter activity well above op0 promoter activity.It could be conclusion that SV40 Enhancer inserted into cow β-casein promoter op0 can significantly improve expression activity of luciferase report gene.更多还原