目的:克隆和鉴定与阿尔兹海默病发生有关的γ-分泌酶的组成蛋白之一nicastrin(NCT)基因的启动子。用于开展其转录调控机制的研究。方法:用PCR方法从人基因组DNA中分离NCT基因编码区上游大小约为1.8kb的片段并以此为基础对启动子进行两端删除分析,分别扩增大小不等的片段,与pGL3-Enhancer荧光素酶报告基因载体质量组,通过瞬时转染Hela细胞,细胞裂解液进行双荧光素酶活性分析,分离NCT基因的启动子区。结果:NCT基因启动子的420bp片段在Hela细胞中具有最强的启动活性,另237bp片段为有活性的最小片段。结论:NCT启动子很可能位于翻译起始位点上游-432/-133处,其基础启动子位于-359/-90。
Objective To identify the promoter of human nicastrin (NCT) gene, a major component of γ-secretase which is closely related with pathogenesis of Alzheimer' s disease. Methods Promoter of human Alzheimer' s disease related gene, nicastrin, a 1 768 bp fragment was firstly isolated from human genomic DNA by PCR. This fragment' s 3 flanking end was 4 bp upstream to the start codon ATG ( + 1 ) of the gene. This fragment was used as template, a series of deleted fragments were amplified and constructed to the pGL3 :Enhancer plasmid with the artificial designed linkers. The relative activity of their promoter in Hela cells was studied by dual-luciferase assay. Results The 420 bp fragment showed the strongest activity, and the 237 bp fragment was the minimal fragment in length with activity. Conclusion The promoter of NCT is located at - 432/-133 region upstream the translational start codon, while its basal promoter is between -359/-90 that drives the transcription of reporter gene in Hela cells.