人TIPE2基因启动子的鉴定及其表达调控区域的研究

Identification of human TIPE2 promoter and its regulatory regions

目的克隆人TIPE2基因启动子区及一系列5'截短片段,并鉴定其DNA表达调控区域。方法采用PCR技术从人基因组DNA中克隆扩增TIPE2基因5'上游1 252 bp启动子序列并对该片段5'端的截短,分别瞬时转染人卵巢癌来源细胞系SKOV3,通过双萤光素酶活性的检测鉴定其启动子活性及表达调控区域。结果克隆扩增的8个TIPE2基因启动子截短片段经测序证明无误;TIPE2-pGL4-F1重组质粒双萤光素酶活性检测显示其具有明显的启动子活性,约为pGL4-Basic的7.9倍;5'系列截短片段重组质粒的启动子活性检测显示,与1 252 bp片段相比,TIPE2上游-965～-593 bp、-501～-390 bp缺失,启动子活性分别升高6.38、7.82倍;-593～-501 bp的缺失,使-501～+79 bp片段相对于-593～+79 bp片段的启动子活性降低2.04倍。结论人TIPE2基因上游1 252 bp区域有明显的启动子活性,该片段内存在2个潜在的负性调控区和1个潜在的正性调控区。

Objective To identify the promoter region of human tumor necrosis factor-α induced protein 8-like 2（TIPE2）,and important regulatory regions of a 1 331 bp putative TIPE2 promoter.Methods A potential 1 331 bp promoter region and 7 fragments of 5′ truncation upstream TIPE2 gene were amplified from human genomic DNA by PCR.Then they were inserted into the luciferase report gene vector pGL4-Basic and transfected into human SKOV3 cells.Relative luciferase activity was detected to determine promoting activities.Results DNA sequencing results indicated that promoter fragments were correctly identified.Analysis of relative luciferase activity showed that the 1331 bp presented a strong promoter activity,which was increased by 7.9-fold compared with pGL4-Basic.TIPE2 promoter activity was respectively increased by 6.38-fold and 7.32-fold after-965 bp to-593 bp and-501 bp to-390 bp were deleted,while promoter activity was decreased by 2.04-fold after-593 bp to-501 bp was deleted.Conclusion The 1 331 bp region upstream TIPE2 gene has high promoter activity.Two potential negative and one positive regulatory regions were identified by 5′truncation of the 1 331 bp promoter region.