SV40增强子对奶牛β-酪蛋白启动子活性的影响  

Effect of SV40 Enhancer on Cow β-casein Promoter Activity

作　　者：钟宇[1] 邵正[2] 江黎明[3] 

机构地区：[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

出　　处：《中国细胞生物学学报》2013年第2期175-179,187,共6页Chinese Journal of Cell Biology

基　　金：广东省自然科学基金(批准号:5011595)资助的课题~~

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。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 l-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 reoort ene.

关 键 词：SV40增强子 奶牛op0 双荧光素酶报告基因检测系统 

分 类 号：Q78[生物学—分子生物学]