摘要
目的构建人鸟苷酸结合蛋白5(guanylate binding protein 5,GBP5)基因启动子不同长度截短片段的荧光素酶报告基因质粒,通过分析启动子不同截短片段的转录活性,确定其核心调控区域。方法PCR扩增GBP5基因启动子序列,将启动子序列截短为5个不同长度的片段,连接至载体pGL3-basic,构建重组质粒pGL3-GBP5-11/21/31/41/51。将各重组质粒转染至293FT细胞,检测荧光素酶活性;利用JASPAR软件预测GBP5启动子区域的转录因子结合位点,依据预测结果选取靶向核心调控区域的阴阳转录因子1(Yin-Yang transcription factor 1,YY1)进行转录调控活性验证;PCR扩增YY1的CDS序列,构建过表达质粒pIRES2-EGFP-YY1;将pIRES2-EGFP-YY1、pGL3-GBP5-21(-1623~+47 bp)质粒与内参质粒pRL-CMV共转染至293FT细胞,检测荧光素酶活性,分析转录因子YY1对GBP5启动子活性的调控作用。结果菌落PCR、双酶切鉴定证明人GBP5基因启动子报告基因质粒构建正确;JASPAR软件预测GBP5启动子区域存在STAT1、YY1、Foxp3等多个转录因子结合位点;双荧光素酶活性检测结果显示,pGL3-GBP5-21(-1623~+47 bp)启动子活性最高,而pGL3-GBP5-41(-520~+47 bp)启动子活性明显降低,提示GBP5启动子核心区域位于5’UTR上游-1623~-520 bp区域;过表达YY1能明显激活GBP5启动子活性,调控GBP5表达。结论人GBP5基因启动子的核心调控区域位于GBP5启动子5’UTR上游-1623~-520 bp区域,该区域存在转录因子YY1结合位点,YY1过表达能显著影响GBP5启动子活性。
Objective To construct luciferase reporter plasmids of truncated fragments of different lengths of human guanylate binding protein 5(GBP5)gene promoter and analyze the transcriptional activity of each fragment to determine the core regulatory region.Methods GBP5promoter sequence was amplified by PCR,truncated into five fragments of different lengths and connected to pGL3-basic plasmid.The constructed recombinant plasmids pGL3-GBP5-11/21/31/41/51were transfected into 293FT cells and detected for luciferase activity.The binding sites of transcription factors in GBP5promoter region were predicted by JASPAR software,and Yin-Yang transcription factor 1(YY1)targeting the core regulatory region was selected and verified for the transcriptional regulatory activity.The CDS sequence of YY1 was amplified by PCR to construct the overexpression plasmid pIRES2-EGFP-YY1,which was then co-transfected to 293FT cells with plasmids pGL3-GBP5-21(-1623~+47 bp)and internal reference plasmid pRL-CMV,and detected for luciferase activity to analyze the regulation of transcription factor YY1 on GBP5 promoter activity.Results Colony PCR and double enzyme digestion identification proved that the plasmid of human GBP5 promoter reporter gene was correctly constructed;JASPAR software predicted that there were multiple transcription factor binding sites such as STAT1,YY1 and Foxp3 in GBP5promoter region.Double luciferase activity assay showed that pGL3-GBP5-21(-1623~+47 bp)showed the highest promoter activity,while the promoter activity of pGL3-GBP5-41(-520~+47 bp)decreased significantly,suggesting that the core region of GBP5 promoter was located at upstream-1623~-520 bp of 5’UTR;Overexpression of YY1 significantly activated the GBP5 promoter activity and regulated the expression of GBP5.Conclusion The core regulatory region of human GBP5 promoter was located in upstream-1623~-520 bp of the 5’UTR,with a binding site of transcription factor YY1 existing in this region.Meanwhile,overexpression of YY1 significantly effected the activity of GB
作者
叶婷
杨康
王田甜
廖玉娇
杜文倩
黄敏
蒋佩文
李敏惠
杨平
YE Ting;YANG Kang;WANG Tian-tian;LIAO Yu-jiao;DU Wen-qian;HUANG Min;JIANG Pei-wen;LI Min-hui;YANG Ping(不详;School of Basic Medicine,Chengdu Medical College,Chengdu 610500,Sichuan Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2023年第2期138-144,共7页
Chinese Journal of Biologicals
基金
四川省科技厅项目(2022NSFSC0690,23NSFSC1243)
四川省发育与再生重点实验室开放项目(SYS20-06)
国家级大学生创新创业项目(202213705004,202213705001)。
关键词
鸟苷酸结合蛋白5
启动子
荧光素酶报告基因
阴阳转录因子1
Guanylate binding protein 5(GBP5)
Promoter
Luciferase reporter gene
Yin-Yang transcription factor 1(YY1)
