摘要
目的 克隆人卵泡刺激素β亚基（Follicle Stimulating Hormone β-subunit,FSHB）的启动子;构建FSHB启动子的报告基因载体并进行活性分析.方法 设计合成FSHB启动子引物,采用PCR技术从人基因组DNA中扩增FSHB启动子序列;将扩增片段插入载体并利用酶切与测序进行鉴定;亚克隆该基因至PGL6-TA荧光报告基因载体;瞬时转染进Hela细胞,收集细胞裂解液后,用荧光素酶报告基因系统检测报告基因载体的活性.结果 PCR扩增得到人FSHB基因启动子;成功构建pGL6-FSHB-promoter报告基因载体,测序结果表明启动子序列正确;双荧光素酶报告基因检测系统证实构建的报告基因载体具有启动子活性.通过荧光素酶相对表达效率比较,在Hela细胞的体外实验中发现,FSHB-211G→T突变后荧光素酶表达具有更高的效率,差异具有统计学意义（P＜0.05）.结论 成功构建人FSHB基因启动子的克隆及其报告基因载体的构建,为深入研究FSHB转录表达的调控机制提供基础.
Objective To clone follicle stimulating hormone β-subunit （FSHB） promoter,construct the reporter gene vector of FSHB promoter,and analyze the activity.Methods By using PCR,we got the promotor of FSHB from human DNA,then we inserted the amplification products into T vector for sequencing.After sequencing,we put the promoter of FSHB into the pGL6 luciferase reporter vector （pGL6-FSHB-promoter） for luciferase assay.The Luciferase reporter assay system was performed to analyze the activity of pGL6-FSHB-promote vector.Results The promoter of FSHB was obtained by PCR.By using Luciferase reporter assay system,we found that pGL6-FSHB-promote vector had promoter activity.After mutating FSHB-211 G to T,the Luciferase expression had higher efficiency than before （P〈0.05）.Conclusions We cloned human FSHB gene promoter and constructed luciferase reporter vector （pGL6-FSHB-promoter）.This study may be helpful for further investigation about the regulation mechanism of FSHB expression.
出处
《国际医药卫生导报》
2016年第14期2034-2038,共5页
International Medicine and Health Guidance News
基金
南京军区医学科技创新课题（10MA065）
厦门市科技惠民项目（2013220134027）
关键词
多囊卵巢综合征
卵泡刺激素
单核苷酸多态性
荧光素酶表达效率
Polycystic ovary syndrome
Follicle stimulating hormone
Single-nucleotide polymorphism
Luciferase expression efficiency
Polycystic ovary syndrome
Follicle stimulating hormone
Single-nucleotide polymorphism
Luciferase expression efficiency
