中文摘要：

目的克隆人中脑星形胶质来源的神经营养因子MANF（mesencephalic astrocyte-derived neurotrophie factor）基因的启动子区域,并鉴定其转录活性。方法利用TESS在线软件分析MANF基因上游序列中潜在的顺时作用元件。以HepG2细胞的总基因组为模板,通过PCR反应扩增了MANF基因5＇非翻译区1 415 bp片段,并将此片段插入到不含有启动子的pEGFP-1载体中构建了pEGFP-1-MANF-5F重组质粒。用脂质体介导的方法将pEGFP-1-MANF-5F质粒转染到293T细胞,在倒置显微镜下观察绿色荧光的表达。同时,将上述MANF基因5＇非翻译区1 415 bp片段插入到荧光素酶报告基因载体pGL3-Basic中,以构建质粒pGL3-MANF-5F,并通过共转染内参质粒pRL-TK到N2 A细胞中,24 h后检测双荧光素酶的表达。结果重组质粒pEGFP-1-MANF-5F转染293T细胞后,在细胞中可见绿色荧光蛋白;pGL3-MANF-5F重组质粒在N2 A细胞中检测到双荧光素酶的活性,且在Tunicamycin诱导时表达明显升高;结论已成功克隆了MANF基因的5＇端非翻译区,并证实该区域具有启动子活性,且内质网应激可调节MANF的启动子活性,这为进一步研究MANF基因在疾病中的表达调控机制奠定了基础。

英文摘要：

Aim To clone the promoter of human MANF（Mesencephalic astrocyte-derived neurotrophic factor） gene and determine its activity.Methods On-line software TESS was used to predict the potential sites for binding transcription factors in the 5′-flanking of MANF gene.The 5′-flanking of MANF gene was cloned from the genomic DNA of HepG2 cells by PCR and was insert into pEGFP-1 and pGL3-Basic to construct pEGFP-1-MANF-5F and pGL3-MANF-5F,respectively,which were used to determine the activity of promoter of 5′-flanking of MANF gene.The plasmids were transiently transfected into HEK 293T and N2A cell lines by means of lipofectamine.The fluorescence of GFP was observed with a fluorescent microscope.The activity of luciferase was detected with luminometer.Results The plasmids pEGFP-1-MANF-5F and pGL3-MANF-5F were successfully constructed.The fluorescence was observed in HEK 293T cells transfected with pEGFP-1-MANF-5F,but not pEGFP-1 vector.Moreover,the activity of dual-luciferase was markedly increased in the N2A cells transfected with pGL3-MANF-5F,compared with pGL3-Basic.The promoter activity of the 5′-flanking of MANF gene was also up-regulated by endoplasmic reticulum stress inducer tunicamycin.Conclusion The 5′-flanking of MANF gene is successfully cloned and proved to have the activity of promoter,which provides useful materials for the further analysis of the transcriptional regulation of MANF.