人βcatenin启动子的克隆及活性分析

山东大学学报(医学版) ›› 2009, Vol. 47 ›› Issue (11): 21-24.

人βcatenin启动子的克隆及活性分析

张菊¹，关恒云¹，张鹏举¹，陈蔚文¹，刘帅²，尚进²，唐传刚²，姜安丽¹

山东大学 1. 医学院生物化学与分子生物学研究所;
2. 医学院05级六年制临床医学专业，济南 250012

收稿日期:2009-03-26 发布日期:2009-11-16
通讯作者: 姜安丽（1958- ），女，教授，主要从事肿瘤分子生物学研究。 Email:jianganli@sdu.edu.cn
作者简介:张菊（1982- ），女，硕士研究生，主要从事肿瘤分子生物学研究。
基金资助:
国家自然科学基金资助课题（30870732）；山东省自然科学基金资助课题（Y2005C03）。

Cloning human βcatenin gene promoter and analyzing its activity

ZHANG Ju, GUAN Hengyun, ZHANG Pengju, CHEN Weiwen, LIU Shuai, SHANG Jin, TANG Chuangang,　JIANG Anli

1. Institute of Biochemistry and Molecular Biology， School of Medicine，Shandong University， Jinan 250012, China;
2. Grade Five of Clinical Medicine 6 years， School of Medicine， Shandong University, Jinan 250012， China

Received:2009-03-26 Published:2009-11-16

摘要/Abstract

摘要：

目的克隆βcatenin基因5′上游1.8?kb启动子，构建启动子荧光素酶报告基因载体pGL31.8?kb，测定其启动子活性，为进一步研究βcatenin基因表达调控机制奠定基础。方法采用PCR方法从人基因组DNA中扩增βcatenin基因5′上游1.8?kb片段并构建到荧光素酶报道基因pGL3basic载体中，与内参照质粒pRLTk共转染前列腺癌PC3细胞，通过双荧光素酶活性检验测定其启动子活性。结果PCR扩增的1.8?kb片段经测序正确无误；pGL31.8?kb转染PC3细胞48?h后，双荧光素酶活性测定启动子活性（M1／M2）为11.71，是pGL3control活性的2.43倍，为pGL3basic活性的206.31倍,为pGL3promoter活性的21.38倍。结论克隆的βcatenin基因5′上游1.8?kb片段具有较强的启动子活性。

关键词: βcatenin；克隆；启动子；PC3细胞

Abstract:

To clone a 1.8?kb fragment upstream of the βcatenin gene and assay its promoter activity． MethodsA 1.8?kb fragment upstream of the βcatenin gene was amplified by PCR using human genomic DNA as a template． Its promoter activity was determined with dualluciferase reporter assay after it had been cloned into a pGL3basic vector and transfected into PC3 cells． ResultsThe sequence of the 1.8 kb fragment proved to be correct by DNA sequencing． Dualluciferase reporter assay (Ml／M2) was 11.71 at 48?h after PGL31.8?kb was cotransfected with pRLTK into prostate cancer cell PC3 which was about 2.43fold higher than that of pGL3control cotransfection with pRLTK, 206.31 fold higher than that of pGL3basic cotransfection with pRLTK and 21.38 fold higher than that of pGL3promoter cotransfection with pRLTK. ConclusionThe cloned 1.8?kb fragment upstream of the βcatenin gene presented strong promoter activity．

Key words: βcatenin gene； Cloning； Promoter; PC3 cellβ

中图分类号:

Q344.13

张菊1，关恒云1，张鹏举1，陈蔚文1，刘帅2，尚进2，唐传刚2，姜安丽1. 人βcatenin启动子的克隆及活性分析[J]. 山东大学学报(医学版), 2009, 47(11): 21-24.

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