激活蛋白2家族基因真核表达载体的构建及其功能鉴定

doi:10.16352/j.issn.1001-6325.2022.12.1829

基础医学与临床 ›› 2022, Vol. 42 ›› Issue (12): 1829-1834.doi: 10.16352/j.issn.1001-6325.2022.12.1829

激活蛋白2家族基因真核表达载体的构建及其功能鉴定

米月, 蒋琦炜, 沈雁婕, 张德宇^*, 叶棋浓^*

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

收稿日期:2021-12-28 修回日期:2022-05-26 出版日期:2022-12-05 发布日期:2022-11-23
通讯作者: ^* yeqn66@yahoo.com;ggatsby@126.com
基金资助:
国家自然科学基金(81930078)

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

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

Received:2021-12-28 Revised:2022-05-26 Online:2022-12-05 Published:2022-11-23
Contact: ^* yeqn66@yahoo.com;ggatsby@126.com

摘要/Abstract

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。

关键词: 人AP-2基因家族, 肝癌, 真核表达, HepG2细胞系

Abstract: 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-2β and AP-2γ on proliferation and migration of HepG2 cell line. Methods Ap-2α,AP-2β and AP-2γ gene 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/γ was 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/γ were 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-2β and AP-2γ may inhibit the proliferation and migration of HepG2 cells.

Key words: human AP-2 family genes, hepatocellular carcinoma, eukaryotic expression, cell line HepG2

中图分类号:

R735.7

米月, 蒋琦炜, 沈雁婕, 张德宇, 叶棋浓. 激活蛋白2家族基因真核表达载体的构建及其功能鉴定[J]. 基础医学与临床, 2022, 42(12): 1829-1834.

MI Yue, JIANG Qi-wei, SHEN Yan-jie, ZHANG De-yu, YE Qi-nong. Construction and functional verification of AP-2 gene family eukaryotic expression vector[J]. Basic & Clinical Medicine, 2022, 42(12): 1829-1834.

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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

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