β_1整合素启动子荧光素酶报告基因载体的构建与鉴定

Construction and identification of β_1 integrin promoter-luciferase reporter gene recombinant vectors

目的克隆β1整合素(CD29)启动子区基因全长序列,构建并鉴定β1整合素启动子全长序列荧光素酶报告基因载体pGL3-β1。方法以含β1整合素启动子基因全长序列的基因片段(2 735 bp)的重组pGEM-T easy载体为模板,用含有酶切位点的特异性引物扩增出整合素β1启动子基因全长序列1 756 bp,克隆至荧光素酶表达载体pGL3-basic,构建含正确目的基因的表达载体pGL3-β1,并NheⅠ、XhoⅠ酶切、PCR及测序鉴定;并以该质粒转染人表皮细胞株HaCaT进行活性检测。结果酶切、PCR及序列测定表明,克隆获得的1 756 bp与GenBank DNA序列数据库对比分析序列一致,插入方向正确;且pGL3-β1启动子在人表皮细胞株HaCaT中有明显转录活性。结论成功构建了β1整合素启动子基因全长序列荧光素酶报告基因载体,为下一步研究人β1整合素启动子的活性分析、基因表达调控机制及其信号转导通路等研究奠定基础。

Objective To clone full length of β1 integrin gene promoter, and to construct and identify β1 integrin gene promoter- pGL3-β1 vectors. Methods Recombinant of pGEM-T easy vector （2 735 bp） including β1 integrin gene promoter full sequence was used as a template in the polymerase chain reaetion（PCR） to amplify its promoter sequence（ 1 756 bp）. The PCR product was directly cloned into the luciferase reporter vector pGL3-Basic. Then the products were identified by DNA sequencing, nest PCR and restriction enzyme digestion. Then it was transfected into HaCaT cells, and the luciferase activity of the cells was analyzed after transfection. Results Restriction enzyme digestion and DNA sequencing confirmed that the sequenced segment（1 756 bp） in the recombinant was identical to that in GenBank and the segment was inserted in right direction. The activity of pGL3-β1 was significantly higher than that in pGL3-basic vectors. Conclusion The luciferase expression vector-pGL3-β1 containing β1 integrin gene promoter full length sequence is constructed successfully. The construction of pGL3-β1 recombinant plasmid lay a foundation of the analysis of prorooter activities, gene expression regulatory mechanism and signal transduction of β1 integrin.