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人甘丙肽2型受体(GalR2)作为G蛋白偶联受体,其功能已被广泛研究,但GalR2基因的转录调控机制尚不明确。我们利用聚合酶链式反应(polymerase chain reaction,PCR)方法扩增人GalR2基因上游1121 bp的片段,再通过PCR方法将其缺失成3段长度不等的片段,然后分别将其克隆到荧光素酶报告基因载体pGL3-Basic中,并将相应的质粒命名为pGL3-B-1121、pGL3-B-922、pGL3-B-521和pGL3-B-200;通过荧光素酶活性分析检测不同长度的GalR2基因启动子在人神经母细胞瘤细胞(SK-N-SH)中的活性,发现GalR2基因上游1121 bp区段具有明显的转录活性,缺失-1121到-922 bp区域引起启动子活性的明显增高,暗示该区域存在负调控元件;利用TESS在线软件分析GalR2基因上游序列,发现该基因启动子含有Sp1、AP-1、AP-2、NF-1、YY1和ERα等多种顺式作用元件。因此,本实验初步确定了人GalR2基因启动子的转录调节区,为进一步研究该基因转录调控机制奠定了基础。
The function of human galanin 2 receptor (GalR2) as a G-protein coupled receptor has been extensively studied, but the mechanism of transcriptional regulation of GalR2 gene is not yet clear. We amplified the 1121 bp fragment upstream of the human GalR2 gene by polymerase chain reaction (PCR) and then deleted it into three fragments of different lengths by PCR, and then cloned them into luciferase PGL3-B-922, pGL3-B-521 and pGL3-B-200, respectively. The luciferase activity assay was used to detect the expression of different length The activity of GalR2 gene promoter in human neuroblastoma cells (SK-N-SH) showed that the 1121 bp upstream of GalR2 gene had obvious transcriptional activity. The deletion of -1121 to -922 bp region caused obvious promoter activity Which indicates that there are negative regulatory elements in this region. Using the software of TESS to analyze the sequence of GalR2 gene, we found that the promoter of the gene contains many cis-acting elements such as Sp1, AP-1, AP-2, NF-1, YY1 and ERα . Therefore, we initially determined the transcriptional regulatory region of human GalR2 promoter, which lays the foundation for further study on the transcriptional regulation of this gene.