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中国癌症防治杂志

中国癌症防治杂志 ›› 2024, Vol. 16 ›› Issue (5): 586-592.doi: 10.3969/j.issn.1674-5671.2024.05.12

• 基础研究 • 上一篇    下一篇

AGK基因启动子重组质粒构建及其在T淋巴细胞白血病中转录活性的研究

  

  1. 中国医学科学院北京协和医学院医药生物技术研究所微生物代谢工程研究室；首都医科大学基础医学院；军事科学院军事医学研究院生物工程研究所
  • 出版日期:2024-10-25 发布日期:2024-11-06
  • 通讯作者: 徐小洁 E-mail：miraclexxj@126.com； 车永胜 E-mail：ysche@imb.cams.cn
  • 基金资助:
    国家重点研发计划（2023YFA0914900）；国家自然科学基金青年科学基金项目（82404651）；北京市自然科学基金?昌平创新联合基金项目（L234042）

Construction of recombinant plasmid of AGK gene promoter and its transcriptional activity in T lymphocyte leukemia

  • Online:2024-10-25 Published:2024-11-06

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摘要： 目的 研究T淋巴细胞白血病中酰基甘油激酶（acylglycerol kinase，AGK）上游转录机制，构建AGK基因启动子不同截短片段的重组质粒并检测其转录活性。方法 利用TIMER2.0、The Human Protein Atlas、Gene Expression Profiling Interactive Analysis等公共数据库探索AGK在白血病中的作用，以pGL4.20⁃pAGK promoter⁃luciferase全长质粒为模板，利用PCR法分别扩增AGK启动子不同长度的截短片段；将扩增片段分别插入pGL4.20⁃basic载体，构建AGK启动子不同截短片段重组质粒；经双酶切及序列鉴定正确后，将重组质粒电击转染至人T淋巴细胞白血病细胞Jurkat中，采用双荧光素酶报告基因实验测定AGK启动子不同截短片段的双荧光素酶活性。结果 AGK在多种类型肿瘤中表达上调（P<0.05），且在白血病细胞中处于较高水平。Log⁃rank检验结果显示AGK高表达患者的总生存期短于低表达患者（P=0.028）。JASPAR网站预测出3个评分高的转录因子TEAD与AGK的结合区域，并成功构建含AGK基因启动子不同截短片段重组质粒。双荧光素酶报告基因实验发现T淋巴细胞白血病细胞中AGK启动子转录活性在-540 bp至-80 bp区域较高。结论 人T淋巴细胞白血病细胞中AGK基因启动子在-540 bp至-80 bp区域的转录活性较高，可为进一步研究T淋巴细胞白血病中AGK的转录及其上游调控机制奠定基础。

关键词: T淋巴细胞白血病, 酰基甘油激酶, 转录因子, 重组质粒, 双荧光酶报告基因

Abstract: Objective To investigate the upstream transcription mechanism of acylglycerol kinase (AGK) in T lymphocyte leukemia, construct recombinant plasmids with different truncated fragments of the AGK gene promoter and detect their transcriptional activity. Methods Public databases such as TIMER2.0, The Human Protein Atlas, and Gene Expression Profiling Interactive Analysis were used to explore the role of AGK in non⁃solid tumors. Using the pGL4. 20⁃pAGK promoter luciferase full⁃length plasmid as a template, PCR was used to amplify truncated fragments of different lengths of the AGK promoter. The amplified fragments were inserted into the pGL4. 20⁃basic vector respectively to construct recombinant plasmids with different truncated fragments of the AGK promoter. After double enzyme digestion and sequence identification were correct, the recombinant plasmid was electroporated and transfected into human T lymphocytic leukemia cell Jurkat, and the dual⁃luciferase reporter gene assay was used to determine the dual luciferase activity of different truncated fragments of the AGK promoter. Results The expression of AGK was up⁃regulation in a variety of tumor types (P<0.05), and the expression level of AGK in leukemia cells was at a higher level. Log⁃rank test showed that the overall survival of patients with high AGK expression was lower than that of patients with low AGK expression (P=0.028）. Three high⁃scoring binding regions of transcription factor TEAD and AGK were predicted by JASPAR website, and the recombinant plasmids containing different truncated fragments of the AGK gene promoter were successfully constructed. Dual luciferase reporter gene assay showed that the transcriptional activity of AGK promoter was higher in the -540 bp to -80 bp region in T lymphocyte leukemia cells. Conclusions The transcription activity of the AGK gene promoter in the -540 bp to -80 bp region was higher in human T lymphocytic leukemia cells, laying the foundation for further research on the transcription and upstream regulatory mechanisms of AGK in T lymphocyte leukemia.

Key words: T lymphocyte leukemia, Acylglycerol kinase, Transcription factor, Recombinant plasmids, Luciferase reporter gene

中图分类号: 

  • R733.7