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  

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

作　　者：彭明玉 何庆玲 王文博 赵聃[1] 张婧[1] 王迎伟[1] 邱文[1] PENG Mingyu;HE Qingling;WANG Wenbo;ZHAO Dao;ZHANG Jing;WANG Yingwei;QIU Wen(Department of Immunology,Nanjing Medical University,Nanjing 211166,China)

机构地区：[1]南京医科大学免疫学系,江苏南京211166

出　　处：《南京医科大学学报（自然科学版）》2021年第5期637-642,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(31470853,31770934,81971468)。

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p IRES2-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p IRES2-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。Objective:This study aims to construct luciferase reporter plasmids of rat S100 calcium binding protein A8(S100 A8) gene promoter and detect their activity in HEK293 T cells in response to SRY-box transcription factor 7(SOX7)overexpression,meantime screen the possible binding elements for SOX7.Methods:Rat S100 A8 gene full length promoter was amplified by PCR and cloned into the luciferase reporter plasmid(pGL3 basic),and named pGL3 S100 A8 FL.The plasmid of pGL3 S100 A8 FL and previously constructed plasmid of pIRES2-SOX7 were co-transfected into HEK293 T cells,and then the luciferase activity was detected.Meanwhile,the potential SOX7-binding elements within S100 A8 promoter were predicted by JASPAR.Based on the predicted results,four plasmids of truncated S100 A8 gene promoter(pGL3 S100 A81~4)were co nstructed.The plasmids of pGL3 S100 A8 FL or pGL3-S100 A81~4 and pIRES2 SOX7 were cotransfected into HEK293 T cells respectively.Then,the luciferase activity was detected.Next,S100 A8 gene promoter of SOX7-binding element(-86~-57 nt)mutated plasmid was constructed(pGL3-S100 A8-M).The HEK-293 T cells were transfected with pGL3-S100 A8-M and pIRES2-SOX7 plasmid,and the luciferase activity was detected.Results:The plasmids of p GL3 S100 A8 FL and pIRES2 SOX7 were co transfected into HEK293 T cells,found that the luciferase activity of S100 A8 gene promoter was markedly increased in response to SOX7 overexpression.The plasmids of pGL3 S100 A8 FL or pGL3 S100 A81~4 and pIRES2 SOX7 were co transfected into HEK293 T cells,and the result displayed that the activity of pGL3 S100 A8-4 was much lower than that in p GL3 S100 A8-FL and pGL3-S100 A8-1~3,indicating that the region of rat S100 A8 promoter(-200~+51 nt)might contain a SOX7-binding element(-86~-57 nt).Then the-86~-57 nt mutated plasmid(pGL3 S100 A8 M)or pGL3 S100 A8 FL and pIRES2 SOX7 were co transfected into HEK293 T cells,and the result revealed that the activity of pGL3 S100 A8-M was much lower than that of pGL3 S100 A8 FL,indicating that the SOX7 may bind to the

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 

分 类 号：R392.12[医药卫生—免疫学]