CAJ | 학술논문

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。
목적：구건함유불동장도인FBXO31기인계동자편단적형광소매보고기인재체，통과검측형광소매활성조도전록활성최강적계동자구역。방법：통과UCSC재선연건분석FBXO31기인계동자서렬。종정상인혈액기인조DNA중확증불동장도FBXO31기인계동자편단，장저사계동자편단삽입pGL3-basic형광소매보고기인재체，구건pGL3-FBXO31-I，pGL3-FBXO31-Ⅱ，pGL3-FBXO31-Ⅲ중조재체。이용지질체개도적전염기술장저3개중조재체분별도입HEK293，A549화HepG2세포，검측형광소매활성，학정 FBXO31계동자전록활성。결과：중조재체적형광소매활성유강지약배렬순서위：pGL3-FBXO31-I，pGL3-FBXO31-Ⅲ，pGL3-FBXO31-Ⅱ。결론：pGL3-FBXO31-I중조재체중함유적FBXO31계동자편단전록활성최강。학정FBXO31계동자전록활성최강구역，위천명FBXO31기인적전록조공궤제전정료견실적기출，위운용FBXO31계동자화특이성파세포치료각충유전성질병제공료이론의거。
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 cels 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.