激活蛋白2家族基因真核表达载体的构建及其功能鉴定  

Construction and functional verification of AP-2 gene family eukaryotic expression vector

作　　者：米月 蒋琦炜 沈雁婕 张德宇 叶棋浓 MI Yue;JIANG Qi-wei;SHEN Yan-jie;ZHANG De-yu;YE Qi-nong(Department of Medical Molecular Biology,Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]军事科学院军事医学研究院生物工程研究所细胞工程研究室,北京100850

出　　处：《基础医学与临床》2022年第12期1829-1834,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(81930078)。

摘　　要：目的构建激活蛋白2(AP-2)家族真核表达载体,探究AP-2α、AP-2β、AP-2γ对人肝癌细胞系HepG2增殖及迁移的影响。方法模板为人卵巢文库,通过PCR扩增AP-2α、AP-2β以及AP-2γ,并与载体pCDNA3.0-FLAG连接得到真核表达载体pCDNA3.0-FLAG-AP-2α/β/γ;通过CCK8法、克隆形成实验和划痕实验分别验证AP-2α、AP-2β以及AP-2γ对肝癌HepG2细胞增殖和迁移的影响。结果成功构建了AP-2基因家族主要成员AP-2α、AP-2β、AP-2γ的真核表达载体,经酶切及基因测序鉴定重组质粒AP-2家族转录因子构建成功,并且能够成功表达;同时经过功能鉴定,显示AP-2家族蛋白可以显著抑制HepG2细胞的增殖以及迁移(P<0.05)。结论构建的AP-2家族基因真核表达载体转染至HepG2细胞后,明显抑制HepG2细胞增殖和迁移。Objective To construct the eukaryotic expression recombinant plasmids of activator protein 2(AP-2)family genes and to identify the effect of AP-2α,AP-2βand AP-2γon proliferation and migration of HepG2 cell line.Methods Ap-2α,AP-2βand AP-2γgene were amplified with human mammary cDNA library as template and ligated with pCDNA3.0-FLAG vector,respectively.The eukaryotic expression vector pCDNA3.0-FLAG-AP-2α/β/γwas constructed and transfected into HepG2 cells.CCK8 assay and clone formation assay were used to detect the effect of AP-2 family genes on HepG2 cell proliferation.Scratch assay was used to detect the effect of AP-2 family genes on HepG2 cell migration.Results The pCDNA3.0-FLAG-AP-2α/β/γwere successfully constructed and expressed in HepG2 cells.AP-2 family genes significantly inhibited the proliferation and migration of HepG2 cells(P<0.05).Conclusions Ap-2α,AP-2βand AP-2γmay inhibit the proliferation and migration of HepG2 cells.

关 键 词：人AP-2基因家族 肝癌 真核表达 HEPG2细胞系 

分 类 号：R735.7[医药卫生—肿瘤]