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小鼠Lrp5基因5′端调控区域的功能

Functional Analysis of 5′ Upstream Control Region of Mouse Lrp5 Gene
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摘要 为研究小鼠低密度脂蛋白(LDL)受体相关蛋白5(LRP5)基因5′端调控序列的功能,PCR扩增小鼠Lrp5基因翻译起始位点上游3041bp(-2909bp^+132bp)DNA序列.PCR产物定向克隆到pGL3-basic载体上,重组质粒命名为pGL3-2909.以pGL3-2909质粒为模板,以不同的引物扩增出不同长短的DNA片段,分别定向克隆到含小鼠Lrp5基因基本启动子并含有荧光素酶报道基因的pGL3-103载体上,构建了12种荧光素酶报告基因表达体系:pGL3-267,pGL3-513,pGL3-535,pGL3-560,pGL3-575,pGL3-623,pGL3-645,pGL3-719,pGL3-770,pGL3-1032,pGL3-1330,pGL3-1619.以pRL-TK为内参照质粒,瞬时转染COS-7细胞,48h后收集细胞测定荧光素酶相对表达活性,pGL3-575(-2909bp^-2334bp)活性是pGL3-513(-2909bp^-2396bp)的20%,pGL3-535(-2909bp^-2374bp)的活性是pGL3-513的44%,pGL3-575的活性是pGL3-560(-2909bp^-2349bp)的48%,均有显著性差异.结果表明,在-2396bp与-2374bp之间的22bp区域内以及-2349bp与-2334bp之间的15bp区域内存在负调控元件.软件分析表明,此区域含有IK2,LYF1及MZF1调控元件. To identify the functional elements of 5′ upstream control region of mouse LDL receptor related protein 5 （LRPS） gene, the upstream region 3 041 bp （-2909 bp - ＋ 132 bp） from the translation initiation eodon of mouse Lrp5 gene was amplified by PCR. The PCR product was cloned into the lueiferase reporter vector pGL3-basie, and the recombinant plasmid pGL3-2 909 was constructed. The different upstream regions were amplified using plasmid pGL3-2 909 as a template, thereafter inserted into the pGL3-103 vector which harbored Lrp5 gene basal promoter and lueiferase reporter gene. Twelve lueiferase expression vectors containing 5′ upstream regions of mouse Lrp5 gene were constructed, including pGL3-267, pGL3-513, pGL3- 535, pGL3-560, pGL3-575, pGL3-623, pGL3-645, pGL3-719, pGL3-770, pGL3-1 032, pGL3-1 330 and pGL3- 1 619 individually. All expression vectors were transfeeted into COS-7, respectively. Relative lueiferase activities in each cell lysate were measured after 48 hours. The relative lueiferase activity of pGL3-575 （ - 2 909 bp- - 2 334 bp） was 20% of pGL3-513 （ - 2 909 bp - - 2 396 bp）, the activity of pGL3-535 （ - 2 909 bp - - 2 374 bp） was 44% of the pGL3-513 and pGL3-575 was 48% of pGL3-560 （ - 2 909 bp - -2 349 bp）. The results suggest that the negative control elements existed in the 22 bp region between the -2396bpand -2374 bp, as well as the 15 bp region between the -2 349 bp and -2 334 bp. IK2, LYF1 and MZF1 consensus sequences were identified using Mat Inspector V2.2 soft ware.
作者 吕萍 龚瑶琴 李江夏 周海斌 陈丙玺
机构地区 山东大学医学院医学遗传学研究所
出处 《中国生物化学与分子生物学报》 CAS CSCD 北大核心 2005年第5期616-621,共6页 Chinese Journal of Biochemistry and Molecular Biology
基金 教育部科学技术研究重点项目(No.02129)资助~~
关键词 LDL受体相关蛋白5 小鼠 Lrp5基因 调控序列 荧光素酶分析 LDL receptor related protein 5, mouse, Lrp5 gene, control region, luciferase activity
分类号 R394 [医药卫生—医学遗传学] Q78 [医药卫生—基础医学]
  • 相关文献

参考文献10

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二级参考文献16

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中国生物化学与分子生物学报
2005年 第5期

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