摘要
目的对人β位点裂解酶-1(BACE1)基因核心启动子进行克隆,构建携带BACE1基因启动子的荧光素酶报告载体,筛选稳定表达细胞株并分析其转录活性。方法提取人胚肾HEK293细胞基因组DNA,以其为模板,PCR扩增BACE1核心启动子(-691^+67)并克隆至荧光素酶报告载体pGL4.21中,构建BACE1基因启动子荧光素酶报告载体pGL4.21-BACE1,将其转染HEK293细胞(无启动子的pGL4.21载体作阴性对照),利用嘌呤霉素筛选稳定表达株后检测其转录活性。结果成功扩增到758bp的BACE1核心启动子,pGL4.21-BACE1载体经双酶切鉴定正确;HEK293细胞被该载体转染后经嘌呤霉素筛选得到6株稳定表达BACE1启动子的细胞株,其转录活性分别是对照组(HEK293/pGL4.21)的(134.7±22.3)、(634.0±13.9)、(437.6±6.1)、(805.5±5.5)、(492.8±59.1)、(1 021.1±46.6)倍(P=0.001)。结论成功构建了人BACE1基因启动子荧光素酶报告载体。
Objective To clone humanβ-site APP cleaving enzyme（BACE1）gene core promoter for constructing luciferase reporter gene vector carrying BACE1 gene promoter and screening stable expression cell line,and to investigate its transcriptional activity.Methods The human embryo kidney HEK293 cell genome DNA was extracted as the template,BACE1 core promoter（-691~＋67）was amplified by PCR,then was inserted into luciferase reporter vector pGL4.21.BACE1 gene promoter luciferase reporter vector pGL4.21-BACE1 was constructed,which was transfected into HEK293cell（pGL4.21 vector without promoter as the negative control）,after screening stable expression cell line by puromycin,the transcriptional activity was detected.Results About758 bp BACE1gene core promoter was successfully amplified by PCR.pGL4.21-BACE1 vector was correct by double enzyme identification.After transfecting HEK293 cell by this vector,6cell strains stably expressing BACE1 promoter were obtained,and their transcriptional activities were（134.7±22.3）,（634.0±13.9）,（437.6±6.1）,（805.5±5.5）,（492.8±59.1）,（1 021.1±46.6）times（P=0.001）of the control group（HEK293/pGL4.21）respectively.Conclusion Luciferase reporter vector of BACE1 gene promoter is constructed successfully.
出处
《重庆医学》
CAS
北大核心
2017年第3期302-304,共3页
Chongqing medicine
基金
国家自然科学基金国际合作重大项目(81220108010)
国家自然科学基金(81171197)
重庆市卫生局重点基金(2011-1-018)
关键词
β位点裂解酶-1
核心启动子
荧光素酶报告载体
高通量药物筛选
β-site APP cleaving enzyme-1
core promoter
luciferase reporter gene vector
high-throughput drug screening
