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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

27卷

期数:

2021年04期

页码:

413-418

栏目:

技术与方法

出版日期:

2021-07-20

文章信息/Info

Title:

Construction of NNMT promoter dual luciferase reporter system and verification of its targeting relationship with SND1

文章编号:

1006－8147（2021）04-0413-06

作者:

卢鑫¹; 2; 任媛媛³; 葛林³; 杨洁³; 何津岩⁴; 付晓³

（1.天津医科大学基础医学院细胞生物学系，天津 300070；2.天津医科大学总医院检验科，天津 300052；3.天津医科大学基础医学院生物化学与分子生物学系，天津 300070；4.天津医科大学基础医学院生理学与病理生理学系，天津 300070）

Author(s):

LU Xin¹; 2; REN Yuan-yuan³; GE Lin³; YANG Jie³; HE Jin-yan⁴; FU Xiao³

（1.Department of Cellular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China；2. Clinical Laboratory, General Hospital，Tianjin Medical University，Tianjin 300052，China；3. Department of Medical Biochemistry and Molecular Biology，School of Basic Medical Sciences，Tianjin Medical University，Tianjin 300070，China；4. Department of Physiology and Pathophysiology，School of Basic Medical Sciences，Tianjin Medical University，Tianjin 300070，China）

关键词:

NNMT; SND1; 双荧光素酶报告基因; 免疫共沉淀; 蛋白相互作用

Keywords:

-

分类号:

Q784

DOI:

-

文献标志码:

A

摘要:

目的：构建尼克酰胺-N-甲基转移酶（NNMT）启动子区的双荧光素酶报告质粒并检测其活性，从而验证SND1蛋白对NNMT基因的调控作用。方法：根据NNMT基因启动子区上、下游-1 500至+200区间的序列设计引物，并以HepG2细胞提取的RNA逆转录得到cDNA为模板进行PCR扩增，得到的NNMT启动子区片段经酶切后连接到双荧光素报告载体GLuc-ONTM启动子报告克隆中，得到Gluc-NNMT 启动子重组质粒，分别与pLVX-IRES-Puro-Flag-SND1质粒和pLVX-IRES-Puro-vector质粒共同转染HeLa细胞，并检测启动子活性。然后用免疫共沉淀的方法验证NNMT蛋白是否与SND1蛋白之间存在相互作用。结果：重组质粒构建成功。双荧光素酶活性实验显示，共转染Gluc-NNMT启动子重组质粒和pLVX-IRES-Puro-Flag-SND1过表达质粒的实验组与对照组相比，NNMT启动子区活性明显增强。结合免疫共沉淀实验结果，证明NNMT与SND1存在相互作用。结论：成功构建了NNMT启动子双荧光素酶报告质粒，SND1对NNMT启动子活性有增强作用，SND1与NNMT蛋白之间存在相互作用。

Abstract:

Objective: To construct a dualluciferase reporter plasmid in the promoter region of Nicotinamide-N-methyltransferase (NNMT) and test its activity，so as to verify the regulatory effect of SND1 protein on the NNMT gene. Methods: Primers were designed according to the sequence from -1 500 to +200 in the upstream and downstream of the NNMT promoter region. PCR amplification was conducted using the cDNA obtained by reverse transcription of RNA extracted from HepG2 cells. The NNMT promoter fragment was ligated with GLuc-ONTM Promoter Reporter Vector after enzymatic digestion，and the Gluc-NNMT promoter recombinant plasmid was obtained.The recombinant plasmid was transfected into HeLa cells with pLVX-IRES-Puro-flag-SND1 plasmid and pLVX-IRES-Puro-vector plasmid，respectively，and promoter activity was detected.Then the interaction between NNMT protein and SND1 protein was verified by Co-immunoprecipitation. Results: The recombinant plasmid was constructed successfully.Dualluciferase activity experiments showed that co-transfection of Gluc-NNMT promoter recombinant plasmid and PLVX-IRES-Puro-flag-SND1 in the experimental group showed significantly increased promoter activity compared to control group.The results of Co-immunoprecipitation showed that NNMT and SND1 had an interaction. Conclusion: NNMT promoter double luciferase reporter plasmid is successfully constructed. SND1 enhances the activity of NNMT promoter，and there is an interaction between SND1 and NNMT protein.

参考文献/References:

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备注/Memo

备注/Memo:

基金项目： 国家自然科学基金(31501056)；天津市教委科研计划项目（2019KJ171）；天津市自然科学基金（18JCYBJC93800）
作者简介：卢鑫（1989-），男，主管技师，硕士在读，研究方向：细胞生物学；通信作者：付晓， E-mail：fuxiao@tmu.edu.cn。

常用功能

更新日期/Last Update: 2021-07-25