| 胡磊 罗文雅 胡威 尤红娟 孔凡运 李向阳 郑葵阳 汤仁仙△.人肝癌细胞LASP1 启动子的克隆及其核心调控区域的初步鉴定[J].,2016,16(4):625-628 |
| 人肝癌细胞LASP1 启动子的克隆及其核心调控区域的初步鉴定 |
| Cloning of the LASP1 Gene Promoter of Human Hepatoma Cells andPreliminary Identification of Its Core Region |
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| DOI： |
| 中文关键词: LASP1 启动子 荧光素酶报告基因 核心区域 |
| 英文关键词: LASP1 Promoter Luciferase reporter gene Core region |
| 基金项目:江苏省高校自然科学基金项目(14KJB310023)；徐州市科技局项目(XZZD1330)；
江苏省脑病生物信息重点实验室开放研究课题(Jsb11401) |
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| 中文摘要: |
| 目的：克隆人肝癌细胞的LASP1 基因的启动子区域并找出该基因启动子的核心调控区域。方法：提取肝癌HepG2 细胞总
DNA，PCR扩增不同长度的LASP1 启动子片段，克隆至PGL3-Basic 载体中构建重组表达载体PGL3-P1(2059 bp)、PGL3-P2(1123
bp)、PGL3-P3(909 bp)、PGL3-P4(574 bp)和PGL3-P5(159 bp)，转化入大肠埃希菌( )DH5琢中，提取质粒经双酶切、PCR 及测序
鉴定阳性克隆并测序。将构建的重组表达载体、PGL3-Basic 载体分别与内参质粒PRL-Tk 共转染HepG2 细胞，48 h后经双荧光素
酶报告基因检测试剂盒检测其活性。结果：PCR结果、双酶切结果以及DNA 测序结果表明成功构建了LASP1 启动子荧光素酶
报告基因载体；双荧光素酶报告基因检测结果显示，与PGL3-Basic 组相比，重组载体PGL3-P1、PGL3-P2、PGL3-P3、PGL3-P4 均具
有较强的启动子活性(P<0.01)，其中，PGL3-P4的活性最强。结论：成功构建了人肝癌细胞不同截断长度的LASP1 基因启动子荧
光素酶报告基因载体，确定了LASP-1 基因启动子的核心区域(-581 bp~-8 bp)，为进一步研究LASP1 基因在肝癌细胞中表达的关
键调节因素及分子机制奠定了基础。 |
| 英文摘要: |
| Objective:To clone the luciferase reporter vectors containing the LASP1 gene promoter of human hepatoma cells and
identify the core regions of the promoter.Methods:The total DNA of HepG2 cells was extracted. Different lengths of the LASP1
promoter were amplified by PCR and cloned into the plasmid PGL3-Basic. Then the constructed recombinant plasmids were transferred
into DH5琢susceptible cells and identified by PCR, enzyme digestion and DNA sequencing. HepG2 cells were co-transfected with
the recombinant expression vectors and internal reference plasmid vectors. The activity of the promoters was evaluated by using dual
luciferase reporter gene assay kit.Results:The results of PCR, double digestion experiments and DNA sequencing showed that the
luciferase reporter vectors of LASP1 promoter were successfully constructed. Compared with the group of PGL3-Basic, the luciferase
activity of the other groups were increased after transiently transfected with PGL3-P1, PGL3-P2, PGL3-P3, PGL3-P4, but not PGL3-P5
(P<0.01). The PGL3-P4 exhibited the maximum luciferase activity.Conclusion:The luciferase reporter vectors of LASP1 promoter of
human hepatoma cells are successfully constructed and the core region (-581 bp~-8 bp) of LASP1 promoter is determined, which
provides a basis for further exploring the key regulatory factor and molecular mechanism of LASP1 expression in hepatoma cells. |
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