小鼠牙本质涎磷蛋白启动子报告基因载体的构建和启动子活性分析

Construction of mouse dentin sialophosphoprotein promoter reporter gene plasmids and analysis of promoter activity of plasmids

目的 :克隆小鼠牙本质涎磷蛋白 ( dentin sialophosphoprotein,DSPP)基因启动子 ,构建含DSPP启动子不同片段的报告基因载体 ,在小鼠成牙本质细胞系 MDPC-2 3中分析各种载体中 DSPP启动子活性。方法 :细胞基因组提取 ,PCR、瞬时转染和报告基因检测。结果 :从 MDPC-2 3细胞基因组中克隆出长为1 .5 kbp的 DSPP启动子 ,将启动子酶切成不同的片断 ,克隆到虫荧光素酶报告基因载体 p GL3 -Enhancer,构建出 4种含 DSPP启动子不同片段的报告基因载体 ,将这些报告基因载体瞬时转染至 MDPC-2 3细胞 ,载体中的启动子具有不同的活性。结论 :成功构建了含小鼠 DSPP启动子片段的报告基因载体 ,为以后研究 DSPP基因表达调控的分子机制提供了实验工具。

AIM:To clone the promoter of mouse dentin sialophosphoprotein (DSPP) gene and construct luciferase reporter gene plasmids containing different DSPP promoter fragments,and to analyze the promoter activity of various constructs in vitro using mouse odontoblast cell line MDPC-23 cells.METHODS:Mouse genomic DNA extraction,PCR,transient transfection and luciferase assay were used.RESULTS:A 1.5-kbp of DSPP promoter was cloned from mouse genomic DNA of MDPC-23 cells. The amplified 1.5-kbp fragment were released by various restriction enzymes and cloned into pGL3-Enhancer luciferase expression vector.The four DSPP promoter luciferase reporter gene constructs had different luciferase activity after transfection into MDPC-23 cells respectively.CONCLUSION:Mouse DSPP promoter luciferase reporter gene constructs were successfully constructed,which will provide a useful tool for study of the molecular mechanism of DSPP expression in future.