人FBXO31基因启动子荧光素酶报告基因载体的构建和活性的检测.docVIP

人FBXO31基因启动子荧光素酶报告基因载体的构建和活性的检测 　　【摘 要】目的：构建含有不同长度人FBXO31基因启动子片段的荧光素酶报告基因载体，通过检测荧光素酶活性找到转录活性最强的启动子区域。方法：通过UCSC在线软件分析FBXO31基因启动子序列。从正常人血液基因组DNA中扩增不同长度FBXO31基因启动子片段，将这些启动子片段插入pGL3-basic荧光素酶报告基因载体，构建pGL3-FBXO31-I，pGL3-FBXO31-Ⅱ，pGL3-FBXO31-Ⅲ重组载体。利用脂质体介导的转染技术将这3个重组载体分别导入HEK293，A549和HepG2细胞，检测荧光素酶活性，确定FBXO31启动子转录活性。结果：重组载体的荧光素酶活性由强至弱排列顺序为：pGL3-FBXO31-I，pGL3-FBXO31-Ⅲ，pGL3-FBXO31-Ⅱ。结论：pGL3-FBXO31-I重组载体中含有的FBXO31启动子片段转录活性最强。确定FBXO31启动子转录活性最强区域，为阐明FBXO31基因的转录调控机制奠定了坚实的基础，为运用FBXO31启动子和特异性靶细胞治疗各种遗传性疾病提供了理论依据。 　　【关键词】FBXO31；启动子；荧光素酶 　　【中图分类号】R749.053 【文献标识码】A 【文章编号】1004-7484（2014）06-3851-03 　　Construction of FBXO31 promoter reporter vector and luciferase activity detection 　　LI Peng YANG La-wei HE Hui-juan CHEN Ting WANG Ya-hong CAI Yan-li HUANG Hai-li* 　　Clinical research center， Affiliated hospital of Guangdong medical college， Zhanjiang， China 524001 　　【Abstract】FBXO31 is a candidate tumor suppressor gene which is located at chromosome 16q24.3. Recent reports indicate that FBXO31 mediates cyclin D1 degradation to induce G1 arrest after DNA damage. There is no report about FBXO31 promoter. The aim of this study is to identify the core reign of FBXO31promoter. We analyzed the reign between upstream 2000bp and downstream 340bp from transcription start point of FBXO31 by using UCSC online tools and identify three possible reigns （FBXO31-I： -1309-+340， FBXO31-II： -600-+284， FBXO31-III： -500-+200）. These reigns was amplified and sub-cloned into pGL3-Basic vector. pGL3-FBXO31-I， pGL3-FBXO31-II， pGL3-FBXO31-III were transfected into HEK293， A549 and HepG2 cells by using lipofectamine 2000. Luciferase activity was detected by using Dual-Luciferase? Reporter Assay. Our results showed pGL3- FBXO31-I possess the strongest activity， which indicate FBXO31-I is the core reign of FBXO31promoter. Our study shed a new light on FBXO31 function. 　　【Keywords】FBXO31； promoter； luciferase 　　前言 　　FBXO31基因是F-box蛋白家族的成员，它位于16号染色体，在人类基因组中的位置为n85920443之间。该基因在基因组中呈逆时针方向排列。FBXO31基因