Page 7 - 南京医科大学学报自然科学版

第41卷第5期 南京医科大学学报（自然科学版）
2021年5月 Journal of Nanjing Medical University（Natural Sciences） ·637 ·

·基础医学·

大鼠 S100A8 基因启动子质粒的构建及其 SOX7 结合元件的初

步鉴定

彭明玉，何庆玲，王文博，赵 聃，张 婧，王迎伟，邱 文 *

南京医科大学免疫学系，江苏 南京 211166

［摘 要］ 目的：构建大鼠S100钙结合蛋白A8（S100A8）基因启动子荧光素酶报告质粒，并在HEK⁃293T中检查过表达性别决
定区 Y 框蛋白 7（SOX7）基因对 S100A8 启动子活性的影响，同时筛选可能的 SOX7 结合元件。方法：采用 PCR 技术扩增大鼠
S100A8 启动子全长，经双酶切后连接到 pGL3⁃basic 中，命名为 pGL3⁃S100A8⁃FL。将 pGL3⁃S100A8⁃FL 与前期构建的 pIRES2⁃
SOX7质粒共转染HEK⁃293T，再测定荧光素酶活性。此外，运用JASPAR预测S100A8启动子区可能包含的SOX7结合元件，并
依此构建4个启动子截短质粒（即pGL3⁃S100A8⁃1~4）。将pGL3⁃S100A8⁃FL和pGL3⁃S100A8⁃1~4分别与pIRES2⁃SOX7共转染
HEK⁃293T，检查荧光素酶活性。接着构建SOX7结合元件突变的S100A8启动子质粒（即pGL3⁃S100A8⁃M），与pIRES2⁃SOX7转
染HEK⁃293T，检测其荧光素酶活性。结果：将pGL3⁃S100A8⁃FL 与pIRES2⁃SOX7共转染HEK⁃293T，发现过表达SOX7可显著
增加 pGL3⁃S100A8⁃FL 启动子活性。将 pGL3⁃S100A8⁃FL 和 pGL3⁃S100A8⁃1~4 分别与 pIRES2⁃SOX7 共转染 HEK⁃293T，发现
pGL3⁃S100A8⁃4 启动子活性显著低于 pGL3⁃S100A8⁃FL 和 pGL3⁃S100A8⁃1~3，提示 SOX7 与 S100A8 启动子结合元件可能位于
-200~+51 nt区域内。将-86~-57 nt元件突变质粒（pGL3⁃S100A8⁃M）或pGL3⁃S100A8⁃FL与pIRES2⁃SOX7共转HEK⁃293T，发现
pGL3⁃S100A8⁃M启动子活性显著低于pGL3⁃S100A8⁃FL。提示SOX7可能与S100A8启动子-86~-57 nt元件结合。结论：成功
构建大鼠S100A8基因启动子全长、截短和突变荧光素酶报告质粒，并初步确定S100A8启动子区的SOX7结合元件。
［关键词］ 性别决定区Y框蛋白7；S100钙结合蛋白A8；启动子；结合元件
［中图分类号］ R392.12 ［文献标志码］ A ［文章编号］ 1007⁃4368（2021）05⁃637⁃06
doi：10.7655/NYDXBNS20210501

Construction of luciferase reporter plasmids of rat S100A8 promoter and initial
identification of SOX7 binding element

PENG Mingyu，HE Qingling，WANG Wenbo，ZHAO Dao，ZHANG Jing，WANG Yingwei，QIU Wen *
Department of Immunology，Nanjing Medical University，Nanjing 211166，China

［Abstract］ Objective：This study aims to construct luciferase reporter plasmids of rat S100 calcium binding protein A8（S100A8）
gene promoter and detect their activity in HEK293T cells in response to SRY ⁃ box transcription factor 7（SOX7）overexpression，
meantime screen the possible binding elements for SOX7. Methods：Rat S100A8 gene full length promoter was amplified by PCR and
cloned into the luciferase reporter plasmid（pGL3basic），and named pGL3S100A8FL. The plasmid of pGL3S100A8FL and previously
constructed plasmid of pIRES2 ⁃ SOX7 were co ⁃ transfected into HEK293T cells，and then the luciferase activity was detected.
Meanwhile，the potential SOX7⁃binding elements within S100A8 promoter were predicted by JASPAR. Based on the predicted results，
four plasmids of truncated S100A8 gene promoter（pGL3S100A8 1~4）were co nstructed. The plasmids of pGL3S100A8FL or pGL3⁃
S100A8 1~4 and pIRES2SOX7 were cotransfected into HEK293T cells respectively. Then，the luciferase activity was detected. Next，
S100A8 gene promoter of SOX7⁃binding element（-86~-57 nt）mutated plasmid was constructed（pGL3⁃S100A8⁃M）. The HEK⁃293T
cells were transfected with pGL3 ⁃ S100A8 ⁃ M and pIRES2 ⁃ SOX7 plasmid，and the luciferase activity was detected. Results：The
plasmids of pGL3S100A8FL and pIRES2SOX7 were co transfected into HEK293T cells，found that the luciferase activity of S100A8
gene promoter was markedly increased in response to SOX7 overexpression. The plasmids of pGL3S100A8FL or pGL3S100A8 1~4 and

［基金项目］ 国家自然科学基金（31470853，31770934，81971468）
∗
通信作者（Corresponding author），E⁃mail：qiuwen@njmu.edu.cn