摘要
目的探讨调控TGF-β1基因高水平转录的启动片段。方法:以人基因组DNA为模板,PCR扩增获得TGF-β1基因转 录起始位点上游5′端-1328～+812bp的片段中不同长度的片段,以此作为启动子与含氯霉素乙酰基转移酶(CAT)报告基因 的质粒pCAT3-enhancer重组成5个重组体,并将其转染至大鼠肝星状细胞株中,测定细胞的CAT的表达水平并比较各重组体 的启动子活性。结果:-308～+812bp序列具有较强的启动活性,-611～+812bp次之,其他几个重组体的启动活性从高到 低的排序依次为-1328～+812bp、-867～+812bp、+227～+812bp。结论:TGF-β1基因启动子片段中,-308～+812bp、 -611～+812bp、-1328～+812bp重组体具有较强的启动活性,是进一步研究人TGF-β1基因转录调控的重要靶序列。
Objective:To investigate the fragments regulating high promoter activity in human transforming growth fac-tor-β 1 (TGF-β 1 )gene.Methods:The fragments of the5′-end of the human TGF-β 1 gene between -1328bp to+812bp were obtained from a healthy male genomic DNA.The target sequences were fused to chloramphenicol acetyltransferase reporter gene in the pCAT3-enhancer plasmid to construct5chimeric recombinant plasmids.These recombinant plasmids were tran-siently transfected into rat hepatic stellate cell line(nFSC)by FuGENE6transfection methods.The putative promoter activity was tested and compared by CAT measurement in transfected cells.Results:The highest CAT expression was demonstrated in construction driven by-308-+812bp,and the CAT activities of-611-+812bp,-1328- +812bp,-867-+812bp dropped down in order,with the lowest CAT expression being+227-+812bp.Conclusion:The fragments of-308-+812bp,-611-+812bp,and-1328-+812bp have higher promoter activity,and they may serve as the target sequences for transcriptional regulation of TGF-β 1 gene. [
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2004年第6期638-641,共4页
Academic Journal of Second Military Medical University
基金
国家自然科学基金(30270605).
