Tet-On系统中多西环素对MBD1诱导表达的调控研究  

Studies on the Regulation of MBD1-induced Expression by Dox in the Tet-On System

作　　者：卢茜 袁月 李丹[1] 张鹏 LU Xi;YUAN Yue;LI Dan;ZHANG Peng(Biology Department of Basic Medical Sciences College,Center for Tissue Engineering and Stem Cell Research,Key Laboratory of Functional Nucleic Acids-Based Biopharmaceutical Research,Guizhou Medical University,Guiyang 550004)

机构地区：[1]基础医学院生物学教研室,组织工程与干细胞实验中心,功能核酸生物药研究重点实验室,贵州医科大学,贵阳550004

出　　处：《生物技术通报》2024年第8期47-52,共6页Biotechnology Bulletin

基　　金：国家自然科学青年基金项目(32000601);贵州省科技计划项目(黔科合基础-ZK[2021]重点005);贵州医科大学青年拔尖人才项目(贵医大优秀青年人才(2020)104号)。

摘　　要：【目的】构建甲基结合蛋白1(MBD1)的Tet-On可诱导表达质粒和293T细胞,探讨多西环素(Dox)对MBD1表达的影响,并通过分析细胞中质粒启动子CpG甲基化水平反映MBD1调控机制。【方法】使用MBD1引物扩增293T的MBD1表达序列,将其连接于Tet-On诱导表达质粒的多克隆位点;将构建成功的质粒用电转染的方法转入MBD1 KO 293T,使用荧光显微镜及Western blot验证获得的MBD1恢复的293T(MBD1 RE 293T);使用不同浓度Dox诱导MBD1表达,利用Western blot检测单克隆细胞中MBD1的表达情况;使用同一浓度Dox诱导MBD1表达,在12、24、48、96、144、192 h收集细胞,利用Western blot检测细胞中MBD1的表达情况;使用亚硫酸氢盐测序方法检测Dox诱导后细胞内可诱导质粒启动子CpG甲基化情况。【结果】成功构建Tet-On诱导表达MBD1质粒;与未诱导细胞相比,Dox诱导后转染细胞MBD1蛋白重新表达;随着Dox浓度的升高细胞中MBD1的表达逐渐增多;相同浓度Dox诱导后,从12-96 h细胞内MBD1的表达逐渐升高,但144 h后MBD1表达下降;细胞内质粒启动子CpG甲基化检测显示,与96 h相比,144和192 h质粒启动子甲基化水平逐渐升高;在诱导144 h加入甲基转移酶抑制剂地西他滨(decitabine)后,质粒启动子甲基化水平降低,MBD1表达升高。【结论】Tet-On可诱导细胞中重新表达MBD1蛋白,并且表达量与Dox浓度正相关,但长时间诱导后细胞内质粒CpG甲基化水平升高,影响细胞中MBD1的表达。【Objective】This work aims to construct the Tet-On inducible expression plasmid of methyl-binding protein 1(MBD1)and 293T cells,to explore the effect of doxycycline(Dox)on the expression of MBD1,and to reflect the regulatory mechanism of MBD1 by analyzing the CpG methylation level of the plasmid promoter in the cells.【Method】The MBD1 expression sequence of 293T was amplified using MBD1 primers and ligated into the polyclonal site of Tet-On inducible expression plasmid.The successfully constructed plasmid was transfected into MBD1 KO 293T by electro-transfection,and the obtained MBD1-restored 293T was verified using fluorescence microscope and Western blot(MBD1 RE).MBD1 expression was induced using different concentrations of Dox,and the expression of MBD1 in monoclonal cells was detected by Western blot.MBD1 expression was induced using the same concentration of Dox,and the cells were collected at 12,24,48,96,144 and 192 h.The expression of MBD1 in the cells was detected by Western blot;and MBD1 expression in the cells was detected by bisulfite sequencing.The expression of MBD1 in the cells was detected by Western blot,and the CpG methylation of inducible plasmid promoter was detected by bisulfite sequencing after Dox induction.【Result】Tet-On inducible MBD1 plasmid was successfully constructed;MBD1 protein was re-expressed in the transfected cells after Dox induction compared with uninduced cells.The expression of MBD1 in the cells increased gradually with the increase of Dox concentration;the expression of MBD1 in the cells increased gradually from 12 to 96 h after induction with the same concentration of Dox,but the expression decreased after 144 h.The expression of CpG methylation of plasmid promoter was detected by bisulfite sequencing method;the CpG methylation of plasmid promoter was detected by bisulfite sequencing method.CpG methylation of the plasmid promoter showed that the methylation level of the plasmid promoter gradually increased at 144 and 192 h compared with that at 96 h.The methylatio

关 键 词：TET-ON MBD1 MBD1ΔCXXC3 甲基化 HEK293T 

分 类 号：Q78[生物学—分子生物学]