人ALOX5AP基因启动子的克隆及生物信息学分析  

Cloning and bioinformatic analysis of human ALOX5AP gene promoter region

作　　者：臧丽玉 徐晟林 耿学峰 姜恩德 吴从平 赵宝昌 ZANG Li-yu;XU Sheng-lin;GENG Xue-feng;JIANG En-de;WU Cong-ping;ZHAO Bao-chang(Shandong First Medical University&Shandong Academy of Medical Sciences,Taian 271016,China)

机构地区：[1]山东第一医科大学(山东省医学科学院),山东泰安271016

出　　处：《泰山医学院学报》2020年第3期183-188,共6页Journal of Taishan Medical College

基　　金：山东省高校科研计划(J18KA142);国家大学生创新创业训练计划(201810439007)。

摘　　要：目的克隆人ALOX5AP(Arachidonate 5-lipoxygenase activating protein)基因启动子区域并构建荧光素酶报告基因表达载体,运用生物信息学分析该序列并预测启动子候选区及转录因子结合位点。方法PCR扩增ALOX5AP基因转录起始点上游2000 bp的片段,将其连接至pGL3-Basic载体,构建荧光素酶报告基因重组质粒,MluⅠ与HindⅢ双酶切及测序鉴定。采用Promoter 2.0、CpGPlot、AliBaba 2.1等软件对启动子及转录因子结合位点进行预测与分析。结果构建了含有ALOX5AP基因启动子序列的重组质粒,双酶切与测序验证构建成功。生物信息学分析表明ALOX5AP基因转录起始点上游2000 bp有3个启动子候选区,未发现CpG岛;上游区域共发现245个潜在的转录因子结合位点,分布在启动子候选区内的有Pit-1α、TBP、TBPWT1-κ、Sox-2等。结论成功构建人ALOX5AP基因启动子荧光素酶报告基因表达载体,生物信息学分析鉴定出启动子候选区,其中含有多个潜在的转录因子结合位点,为ALOX5AP基因的转录调控分析提供了理论依据。Objective:To clone the promoter region of ALOX5AP(Arachidonate 5-lipoxygenase activating protein)gene into a luciferase reporter gene expression vector,and analyze the sequence and predict promoter candidate area and transcription factor binding sites by bioinformatic softwares.Methods:The PCR product of ALOX5AP gene promoter region was connected to the pGL3-basic vector,the recombinant plasmid of lucigenase reporter gene was constructed,and the construction was identified by enzyme digestion and sequenc.Promoters 2.0,CpGPlot,AliBaba 2.1 and other software were adopted to predict and analyze promoters and transcription factor binding sites.Results:A recombinant plasmid containing the promoter sequence of ALOX5AP gene was successfully constructed.Bioinformatics analysis showed that there were 3 promoter candidate regions in 2000 bp upstream of the transcription starting point of ALOX5AP gene,and no CpG island was found.A total of 245 potential transcription factor binding sites were found in the upstream region,some transcription factor binding sites such as Pit-1α,TBP,TBPWT1-κand Sox-2 were found in the candidate regions.Conclusion:The human ALOX5AP gene promoter luciferase reporter gene expression vector is successfully constructed,and the candidate region of the promoter is identified by bioinformatics analysis,which contain multiple potential transcription factor binding sites,providing theoretical basis for the transcriptional regulation analysis of ALOX5AP gene.This result will provide theoretical basis for the transcriptional regulation analysis of ALOX5AP gene.

关 键 词：ALOX5AP基因 脑卒中 启动子 转录调控 

分 类 号：R394.8[医药卫生—医学遗传学]