摘要
目的:克隆急性单核细胞白血病新抗原基因MLAA-34上游启动子序列并检测其活性、鉴定核心区域。方法:PCR扩增MLAA-34基因启动子区全长片段,将其连接至p GL3-Basic载体,克隆重组质粒;构建一系列MLAA-34基因启动子5’侧翼区截短质粒;将含MLAA-34启动子序列的重组质粒及其截短型质粒转染至U937、HEK293细胞,应用双荧光素酶报告基因检测各片段的启动子活性,确定启动子最小活性区域,并通过生物信息学方法分析转录因子结合位点。结果:构建了含有MLAA-34启动子序列的重组质粒及其截短型质粒;与空载体相比,其启动子活性明显增加(P <0. 001)。MLAA-34最小活性区域位于转录起始位点-402 bp至-200 bp之间,其中包含E2F1,MZF-1,SP1,USF2和STAT3等多个转录因子结合位点。结论:成功构建急性单核细胞白血病新抗原基因MLAA-34不同缺失片段的启动子荧光素酶报告基因重组质粒,鉴定出核心启动子区,其中含有多个潜在的转录因子结合序列。
Objective:To clone the promoter sequence of acute monocytic leukemia new antigen gene MLAA-34 and identify its promoter core region.Methods:The full-length fragment of MLAA-34 gene promoter region was amplified by PCR,then was ligated into pGL3-Basic vector,and the recombinant plasmid was cloned.Constructed a series of MLAA-34 gene promoter 5’flanking region truncated plasmid.These recombinant plasmids were transfected into U937 and HEK293 cells,and the dual luciferase reporter gene was used to detect the promoter activity of each fragment to determine the minimum active region.Transcription factor binding sites were analyzed by bioinformatics methods.Results:The recombinant plasmid containing MLAA-34 promoter sequence and its truncated plasmid were successfully constructed,and the promoter activity was significantly increased as compared with the empty vector(P<0.001).The minimal active region of MLAA-34 located between-402 bp and-200 bp.It contained multiple transcription factor binding sites such as E2 F1,MZF4,SP1,USF2 and STAT3.Conclusion:The promoter of luciferase reporter gene has been successfully constructed with different deletion fragments of MLAA-34,and its core promoter region may contain multiple transcription factor sequence.
作者
雷博
张王刚
何爱丽
陈银霞
曹星梅
赵万红
王剑利
刘捷
马肖容
张鹏宇
白菊
LEI Bo;ZHANG Wang-Gang;HE Ai-Li;CHEN Yin-Xia;CAO Xing-Mei;ZHAO Wan-Hong;WANG Jian-Li;LIU Jie;MA Xiao-Rong;ZHANG Peng-Yu;BAI Ju(Department of Hematology,The Second Affiliated Hospital,Medical School of Xi'an Jiaotong University,Xi'an 710004,Shaanxi Province,China)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2019年第3期641-645,共5页
Journal of Experimental Hematology
基金
国家自然科学基金资助(8153000580)
