摘要
目的:HPD是一种酪氨酸分解代谢途径中的关键酶,特异表达于肝脏。目前对HPD的研究主要集中在HPD与酪氨酸血症等疾病的关系上,而对其转录调控的报道较少,并且对HPD在肝脏中特异性表达的转录调控机制尚不清楚,也未见报道。通过构建HPD基因启动子荧光素酶报告基因载体,检测其在肝源细胞中的特异转录活性,进而揭示其肝脏特异性表达的调控机制。方法:运用UCSC在线数据库分析人HPD基因组结构,并获得其5'端上游启动子区域-2000^+39bp的DNA序列。以人的肝癌细胞Hep G2基因组DNA为模板,利用PCR扩增该序列,并克隆到p GL3-Basic荧光素酶报告基因载体上。通过设计不同引物,进一步构建系列缺失体,共获得7个不同长度的荧光素酶报告基因载体。将构建的载体分别转染Hep G2、L02及HEK293细胞,通过双荧光素酶活性分析实验检测不同缺失体的转录活性。结果:报告基因实验结果显示-600^-400区域在肝源细胞系Hep G2和L02细胞中具有较强转录活性,而在HEK293细胞中活性较低,-400^+39区段则在两种细胞中均有转录活性。生物信息学分析结果显示-600^-400区域内存在较多肝脏特异性转录因子结合位点。结论:成功构建了HPD基因启动子荧光素酶报告基因载体,发现-600^-400存在调控HPD肝脏特异表达的关键元件,为进一步深入揭示HPD肝脏特异性表达的转录调控机制奠定了理论基础。
HPD is a kind of tyrosine metabolizing enzyme with specific expression in liver. The current research mainly focuses on the relationship between HPD and tyrosinemia diseases, while there is less reporter about the transcription regulational mechanism of HPD, and the mechanism of HPDliver specific expression is not yet known. The aim was to construct HPD gene promoter luciferase reporter gene vector, analyze its transcriptional activity in the human hepatocellular cells, and begin to clarify the regulational mechanisms of its specific expression in liver. Methods： The 5′flanking sequence from －2 000 to ＋39 of the human HPD gene was analyzed by the UCSC software for the promoter character. This sequence were cloned from the human hepatocellular carcinoma cell HepG2 genomic DNA by PCR and inserted into the pGL3Basic vector.Through the design of different primers, a series of 5′deleted constructs were made to product seven luciferase reporter gene vectors in different lengths. Then the seven reconstructors were transfected into HepG2,L02 and HEK293 cells respectively to determine the transcriptional activity of HPD gene promoter by the dualluciferase analysis.Results： The dualluciferase analysis results showed that the fragment from －600 to －400 had a stronger transcriptional activity in HepG2and L02 than in HEK293,while the fragment from －400 to ＋39 had the transcriptional activity in all of the three cells. Bioinformatics analysis results showed that there are many liver specific transcription factor binding sites in －600 ~－400 area of HPD gene promoter sequence. Conclusion： The luciferase reporter gene vectors with the HPD gene promoters are constructed successfully. In the fragment from －600 to －400,there would have the key regulatory elements for the HPD liverspecific expression,which provides an important basis and lays a theoretical foundation for further revealing the transcriptional regulational mechanism of HPDliver specific expression.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2015年第4期30-41,共12页
China Biotechnology
基金
国家自然科学基金(81472586)
国家"973"计划(2011CB915501)资助项目
关键词
HPD基因
启动子
肝脏特异表达
转录活性分析
Human HPD gene Promoter Liver-specific expression Transcriptional activity analysis
