含人APOBEC3F启动子荧光素酶报告基因载体的构建  

Construction of a luciferase reporter gene vector driven by APOBEC3F promoter

作　　者：赵柯[1] 王临旭[1] 庄严[1] 黄德东[1] 李媛[1] 康文臻[1] 孙永涛[1] 

机构地区：[1]第四军医大学唐都医院传染科,西安710038

出　　处：《传染病信息》2010年第6期333-336,共4页Infectious Disease Information

基　　金：国家自然科学基金(30872347)

摘　　要：目的构建含人载脂蛋白B mRNA编辑酶催化多肽样蛋白3F(apolipoprotein B mRNA editing enzyme,catalytic polypeptide-like3F,APOBEC3F)基因启动子的萤光素酶报告基因载体。方法应用5'cDNA末端快速扩增分析(5'rapid am-plification of cDNA ends,5'RACE)技术确定APOBEC3F转录起始位点,利用PCR技术扩增人APOBEC3F启动子序列,构建含人APOBEC3F启动子的萤光素酶报告基因载体,行双酶切及测序鉴定,并通过双萤光素酶报告基因系统检测启动子活性。结果 APOBEC3F基因转录起始位点位于GenBank已公布的APOBEC3F cDNA起始位置上游13个核苷酸处,测序结果表明克隆获得的APOBEC3F基因启动子序列与GenBank报道一致。重组载体pGL3-A3F-Prom组F/R值(0.342082±0.023516)与空载体pGL3-basic组(0.007871±0.002357)相比,差异有统计学意义(P<0.05)。结论成功构建了含人APOBEC3F基因启动子的萤光素酶报告基因载体,为更深入研究APOBEC3F基因转录调控机制奠定了基础。Objective To construct an apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3F （APOBEC3F） promoter-driven luciferase reporter gene vector. Methods Transcriptional start sites （TSS） were identified by 5＇ rapid amplification of eDNA ends （RACE） analysis, and the promoter of APOBEC3F was amplified via polymerase chain reaction （PCR）. APOBEC3F promoterdriven luciferase reporter gene vector was constructed and verified by double enzyme digestion and sequence analysis. The activity of the promoter was measured by dual-lueiferase reporter gene assay. Results The TSS was located at 13 nueleotides upstream of the start site of APOBEC3F cDNA published in GenBank. Sequence analysis showed that the sequence of cloned APOBEC3F promoter was identical to what C, enBank had reported. There was significant difference in F/R value between pGL3- A3F-Prom vector group （0.342 082_＋0.023 516） and empty vector group （0.007 871＋0.002 357）. Conclusion The lueiferase report gene vector, containing APOBEC3F promoter is constructed successfully, which is a convenient tool to further investigate the mechanism of transcriptional regulation of APOBEC3F.

关 键 词：启动区 遗传 荧光素类 基因 病毒 HIV-1 反转录 

分 类 号：R394-33[医药卫生—医学遗传学] R512.91[医药卫生—基础医学]