一个小型人酪氨酸羟化酶基因启动子的分离和活性分析

目的探讨酪氨酸羟化酶(tyrosine hydroxylase,TH)基因上游调控区的功能。方法PCR法扩增人TH基因上游片段,插入pGL3-Basic质粒,构建一个表达荧光素酶的报告基因。将报告基因瞬间转染MES23.5、293及Cos7细胞,用Dual Luciferase Assay法测定目的DNA片段的启动子活性。结果测序结果表明,分离的DNA片段长度为520bp,与人TH基因-493/+27区一致。与pGL3-Basic质粒相比较,构建的报告基因在3种细胞中显著表达(P<0.01)。结论TH基因-493/+27区具有明显的启动子活性,构建的报告基因有助于研究TH基因表达的调节。

Isolation and Activity Analysis of a Minimal Promoter of the Human Tyrosine Hydroxylase Gene

Objective To investigate the regulatory functions of human tyrosine hydroxylase gene upstream region. Methods A human TH gene upstream fragment was amplified by PCR, and was inserted into plasmid pGL3-Basic, to build a luciferase reporter vector. The reporter vector was transient transfected into MES23.5, 293T and Cos7 cells, and the promoter activity was mensurated using Dual Luciferase Assay system. Results Length of the isolated DNA fragment is 520 bp, which located at -495/+25 region of the human tyrosine hydroxylase gene. By comparison with pGL3-Basic vector, the recombinant reporter vector could significantly expressed luciferase in three types of cells(P<0.01). Conclusion TH gene -493/+27 region possesses promoter activity, and the constructed reporter gene will help us study the regulation of TH gene expression.