人β1整合素启动子核心启动序列初步分析

The Preliminary Analysis of Core Sequences at Human Integrin β1 Promoter

目的:分析人β1整合素在HaCat细胞中核心启动片段。方法:以本室构建的含整合素β1全长启动子1745bp基因序列(-1745bp～+11bp)的重组载体pGL3-1756为模板,用含有酶切位点的特异性引物扩增出整合素β1启动子-845bp～-304bp间基因序列,克隆至荧光素酶表达载体pGL3basic,构建含正确目的基因重组载体pGL3-542。用pGL3-1756、和pGL3-542质粒转染人表皮细胞株HaCat进行活性分析。结果:酶切及序列测定表明,克隆后插入pGL3basic中的启动子片段与GenBank DNA序列数据库对比分析序列一致,且插入方向正确。在人永生化表皮细胞株(HaCat细胞)中构建的pGL3-542载体具有很强的启动活性。结论:成功构建了整合素β1远端启动子542bp片段载体,在HaCat细胞中具有非常强的启动活性。人整合素β1整合素启动子核心启动序列可能位于-845bp～-304bp。

Objective： To analyze the core fragment of integrin betal in HaCat cells. Methods： pGL3-1756, a constructed vector containing the human integrin beta 1 promoter （1756 bp）, was used as template for amplification of the vector, which was named pGL3-542. HaCat cells were transfected with pGL3-1756, promoter sequence at the distal part （-845 bp～-304 bp）. Then the amplified sequence was cloned into pGL3 basic pGL3-542, respectively, for the analysis of promoter activity, and pGL3-basic was used as negative controls. Results： The results of enzyme digestion and sequencing identified the correction of constructed vector, pGL3-542 exhibited very strong activity in HaCat cells. Conclusion： The reporter gene vector containing 542 base pairs at the distal part ofintegrin beta 1 promoter is constructed successfully, with strong activity in HaCat cells. The core sequences at human integrin β1 promoter may be located in -845 bp ～-304 bp.