人XAF1基因启动子的克隆及其在人肝癌细胞株中活性的测定

Human XAF1 gene promoter cloning and assay of its promoter activity in hepatoma cell lines

目的分析XAF1基因启动子在肝癌细胞株中的活性,为研究XAF1基因转录调控的基本机制奠定基础。方法从肝癌细胞株中扩增XAF1基因的1 395 bp启动区域片段并分别克隆到报告基因载体pGL3-basic和pEGFP-1中,分别将含有XAF1基因启动子的报告基因载体pGL3-basic和pEGFP-1转染肝癌细胞株HepG2、SMMC7721,检测萤火虫荧光素酶的活性并观察绿色荧光蛋白的表达。通过转染细胞、荧光素酶活性测定及观察绿色荧光蛋白的表达,检测其启动子活性。结果重组的质粒经双酶切和测序结果证实克隆的XAF1启动子片段序列正确;转染后的HepG2、SMMC7721荧光素酶相对发光强度分别是6.97±0.74、6.12±0.59。其绿色荧光蛋白强度明显低于对照组。结论本实验构建的含XAF1启动子的报告基因质粒为研究XAF1基因的转录调控提供实验依据。

Objective To identify the function of XAF1 gene promoter cloning in hepatoma cell lines. Methods The 1 395 bp fragment of XAF1 gene promoter was amplified by PCR and cloned into pGL3-bastie and pEGFP-1 to examine its activities. The plasmids were transfected into hepatoma cell lines by lipofectamine 2000. The promoter activity was determined by dual-lueiferase report assay and the expression of green fluorescent protein （GEP） reporter was detected by fluorescence microscope. Results The sequence of reeombined plasmids was confirmed by doule restriction and sequencing. The levels of dual-lueiferase reporter activity in HepG2 and SMMC7721 cells were 6.97 ± 0.74 and 6.12 ± 0.59, respectively. And GFP expressions in HepG2 and SMMC7721 ceils were lower than that in control. Conclusion Cloned XAF1 promoter report system has its functional activity.