人短链TSLP启动子区的克隆鉴定及功能分析

Identification and characterization of human short isoform TSLP promoter

目的:构建人短链胸腺基质淋巴生成素(short isoform thymic stromal lymphopoietin,sfTSLP)启动子全长及其长度不等片段的萤光素酶报告质粒,以确定启动子活性区域并预测功能性转录因子结合位点。方法:使用按基因组序列设计的引物,通过PCR分离人sf TSLP启动子片段,并将之插入萤光素酶报告基因载体pGL3-basic中。通过步移缺失构建其截短片段,通过双萤光素酶报告基因系统测定所有启动子缺失克隆在HEK-293细胞中的活性,并使用生物信息学手段预测其潜在的转录结合位点。结果:经菌液PCR、双酶切、测序,成功构建了人sfTSLP启动子及其截短片段萤光素酶报告质粒,并确定其启动子活性区域位于5′侧翼区-200^+25 nt区域内,且可能含有SP1/SP3、SPI1/SPIB、TCF3、ETS1等转录因子结合位点。结论:首次对人sfTSLP启动子区进行了研究,初步确定了其启动子活性区域及可能的转录因子结合位点,为更进一步的研究指明了方向。

Objective:To idefine the regions of the human short isoform thymic stromal lymphopoietin(sfTSLP)promoter and to predict the potential transcription factor binding sites. Methods:Original human promoter fragments were isolated by PCR,using primers generated from genomic sequences of sfTSLP. Amplified fragments were then cloned into the pGL3-basic vector. Five promoter fragments with different length were obtained by walking deletion and cloned into pGL3-basic vector. The vector expression activities were determined by measuring luciferase activity in HEK-293 cells. Its potential transcriptional binding sites were predicted by bioinformatics methods. Results:A nested series of deletion constructs of the human sfTSLP gene promoter were generated and the core promoter region was determined to be located in the-200^+25 region at the 5′ side,which may contain the binding sites of SP1/SP3,SPI1/SPIB,TCF3,ETS1 and other transcription factors. Conclusion:It is the first study on the promoter fragmentsof human sfTSLP. The core promoter region was found and the possible transcription factor binding sites were also preliminarily identified,which can be the basis of further study.