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目的 :分析人CCRK基因启动子、启动子的核心序列及与其表达相关的转录因子。方法 :采用 5′ RACE技术鉴定人CCRK基因的转录起始点 ;对启动子区 3′端 3个截短片段 2 5 5bp(- 36 7/- 12 8) ,2 10bp(- 32 2 /- 12 8)和16 0bp(- 2 72 /- 12 8)进行删除分析 ,双荧光素酶分析测出的最小活性片段作为核心启动子的上游位点。通过生物软件MatInspectorV2 .2分析核心启动子区域 ,寻找并推测重要的转录因子 ,并对其核心序列进行定点突变分析 ,再通过电泳迁移率 (EMSA)实验加以鉴定。结果 :采用 5′ RACE技术 ,得到的PCR产物直接测序 ,定位 - 2 4 2“T”为转录起始点 ;通过启动子 3′端小片段删除分析 ,2 5 5 ,2 10和 16 0bp均无活性 ,由此判断 3′端 2 5 5个核苷酸不存在核心启动子序列 ,并定位一段 74bp的区域为核心启动子 (- 4 4 1/- 36 7)区域。通过MatInspector软件分析 ,发现在这段核心启动子区域内有 3个重要的结合位点 :DeltaEF 1,NF κB和Sp1。对这 3个结合位点的核心序列进行定点突变后瞬时转染U373,经双荧光素酶分析发现DeltaEF 1的活性明显升高 ,NF κB没有变化 ,Sp1的活性降低但不太明显。进一步通过电泳迁移率 (EMSA)实验鉴定 ,DeltaEF 1和NF κB能与U373核蛋白形成特异性的DNA 蛋白质复合体 ,表明De
OBJECTIVE: To analyze the core sequence of human CCRK promoter and promoter and the transcription factors related to its expression. Methods: The 5 ’RACE technique was used to identify the transcription initiation point of human CCRK gene. The 3’ truncated 3 ’ ) And 16 0bp (-272 / - 12 8). The minimal active fragment detected by dual luciferase assay was used as the upstream site of the core promoter. The core promoter region was analyzed by the biological software MatInspector V2.2 to find and speculate the important transcription factors and carry out site-directed mutagenesis analysis of its core sequence, and then identified by electrophoretic mobility shift assay (EMSA). Results: The 5 ’RACE technique was used to directly sequence the PCR products. The locus-2 4 2 “T” was the starting point for transcription. By deletion analysis of the 3’ end of the promoter, none of 2 5 5, 2 10 and 160 bp Thus, it was determined that there was no core promoter sequence at the 235 nucleotides at the 3 ’end and a 74 bp region was located as the core promoter (-414 / 367) region. Analysis by MatInspector software revealed three important binding sites within this core promoter region: DeltaEF 1, NF κB and Sp1. After the site-directed mutagenesis of the three binding sites, U373 was transiently transfected. After double luciferase analysis, the activity of DeltaEF 1 was significantly increased, but there was no change in NF κB, but the activity of Sp1 was decreased but not obvious. Further confirmed by electrophoretic mobility shift assay (EMSA), DeltaEF 1 and NF κB can form specific DNA protein complexes with U373 nucleoprotein, indicating that De