【摘要】 目的:构建能够报告人破骨细胞分化因子(ODF)基因启动子活性的表达载体。方法:以人基因组DNA为模板,PCR扩增含有ODF基因启动子的2段序列(-2433￣-1243bp和-1262￣+100bp),通过T-A克隆将这2个序列片段分别连接到pGEM"-TEasy克隆载体上。利用特定的限制性内切酶位点,将这2个序列片段酶切后进行正确拼接并定向克隆到不含启动子的pEGFP-1报告基因载体上。将该重组质粒pEGFP-1-ODF瞬时转染成骨细胞系UMR106,检测其能否在ODF基因启动子(-2358￣+100bp)的调控下表达报告基因增强型绿色荧光蛋白(EGFP)。结果:pEGFP-1-ODF经酶切鉴定和序列分析证实与设计完全一致。细胞瞬时转染结果表明,pEGFP-1-ODF转染的UMR106能表达EGFP。结论:人ODF基因启动子报告载体的成功构建,为进一步研究ODF基因表达转录水平的调控机制奠定了基础。更多还原

【Abstract】 Objective: To construct a specific reporter vector to monitor the activity of human osteoclast differentiation factor (ODF) gene promoter. Methods: Two DNA segments of ODF gene promoter (-2 433 - -1 243 bp and -1 262 - +100 bp) were amplified by PCR from human genome DNA and cloned into clone vector pGEM-T Easy. The gene fragments were correctly connected to the promoter without vector pEGFP-1 by restriction enzyme. Recombinant plasmid pEGFP-1-ODF was transferred into osteoblastic cell line UMR106. The expression of EGFP was detected under the monitor of human ODF gene promoter (-2 358-+100 bp). Results: pEGFP-1-ODF was the same as the design confirmed by restriction digestion and sequence analysis. The expressing EGFP could be detected in UMR106 by pEGFP-1-ODF transfection. Conclusion: Human ODF gene promoter reporter vector is significant for further study on ODF gene expression transfection level.更多还原