人NaDC1基因近端启动子生物信息学分析及载体构建

Bioinformatics analysis of human NaDC1 proximal promoter and construction of firefly report gene vector

目的对人NaDC1(hNaDC1)基因近端启动子进行生物信息学分析,预测可能的调控区域及顺式元件,并构建相应基因序列萤火虫荧光素酶报告基因表达载体。方法使用First EF程序分析并获得hNaDC1基因近端启动子序列,使用MatInspector5.0软件对近端启动子序列中的转录因子结合位点进行预测。PCR法扩增hNaDC1基因近端启动子序列,PCR产物经KpnⅠ和HindⅢ双酶切后定向克隆到pGL3-Basic载体。重组质粒行KpnⅠ和HindⅢ双酶切及测序鉴定。结果使用FirstEF程序获得长度为2.4kb(-2232/+136)的hNaDC1基因近端启动子序列。MatInspector5.0程序分析显示该基因序列共含有152个74种顺式作用元件。KpnⅠ和HindⅢ双酶切及测序鉴定证实构建的pGL3-hNaDC1A质粒插入片段正确无误。结论成功构建hNaDC1基因近端启动子转录调控序列萤火虫荧光素酶报告基因表达载体,为利用双荧光素酶报告基因检测系统研究hNaDC1基因近端启动子转录调控元件的分布及其性质提供了基本试验条件。

Objective To bioinformatically analyze the human NaDCI promoter for predicting possible regulative zone and cis element and construct firefly luciferase report gene vector for 5＇ flanking regulated elements of hNaDC1 gene. Methods The proximal promoter of hNaDC1 was analyzed by using FirstEF online software to obtain its sequence. Meanwhile, the binding sites of transcription factors were predicted by software Matinspector 5.0. The DNA fragment hNaDC1A （ -2 232/＋ 136） of5＇ flanking of hNaDC1 gene were amplified from the genomic DNAs of human renal proximal tubular cells （HK-2） by PCR, the products of which were cloned into pGL3-Basic after digestion with Kpn Ⅰ and Hind Ⅲ. The recombinant plasmid pGL3-hNaDC1A was used for restriction endonucleases digestion and DNA sequencing. Results The 2.4 kb （ -2 232/＋ 136） sequence of hNaDC1 proximal promoter was obtained by FirstEF. Matinspector 5.0 showed 152 cis-acting elements （74 sorts） within the proximal promoter. The expression vector pGL3-hNaDC1A was constructed successfully. Conclusion The construction of firefly report gene vector offers experimental basis for studying the distribution of transcriptional regulation elements and their characteristics with the help of dual luciferase report gene assay system.