【摘要】 目的:克隆人β2微球蛋白(β2m)基因启动子,并研究其在小鼠肥大细胞瘤细胞P815中对下游报告基因的启动活性。方法:用PCR法从基因组内扩增β2m基因启动子。通过基因重组的方法,构建含此启动子的EGFP基因真核表达载体,然后分别瞬时和稳定转染P815细胞。通过RT-PCR、荧光显微镜摄像和流式细胞术分析,观察IFN-γ作用前后EGFP的表达。结果:从基因组内扩增出302bp的人β2m基因的启动子,成功地构建了含此启动子的真核报告载体。RT-PCR的结果表明,IFN-γ对启动子的活性具有诱导作用,且呈浓度依赖性。流式细胞术的结果显示,在5×105U/LIFN-γ作用下,尽管表达EGFP的细胞数量没有差异,但刺激组EGFP的表达强度是未刺激组的2倍。结论:人β2m基因启动子在小鼠肥大细胞瘤细胞P815中具有高度的启动活性。通过该启动子中所含干扰素刺激反应元件(ISRE),可在IFN-γ诱导作用下调控下游目的基因的表达。更多还原

【Abstract】 AIM: To clone the human β2-microglobulin(β2m)gene promoter and study its activity in P815 cells. METHODS: PCR amplification was performed using primers based on human β2m gene sequence from GenBank and human genomic DNA as a template. The PCR product was directedly ligated into pBluescriptⅡ vector for sequencing. The promoter fragment was subcloned into a pcDNA3-EGFP plasmid after it had been idendified correctly. A mouse mastocytoma cell line P815 was transiently and stably transfected with the plasmid containing human β2m gene promoter and enhanced green fluorescence protein(EGFP) gene. The mRNA expression of EGFP in transiently tranfected cells was quantified by RT-PCR and that in stably transfected cells was detected by fluorescence microscope and flow cytometry(FCM). RESULTS: A 302 bp DNA fragment was amplified and cloned into the pcDNA3-EGFP vector. RT-PCR analysis showed that EGFP mRNA expression was induced by IFN-γ in a dose-dependent manner. There was no difference in the fluorescence positive cell rate between the IFN-γ-treated group and the control group. But the fluorescence intensity of the 5×10~5 U/L IFN-γ-treated group increased about 2 folds compared with that of the control group. CONCLUSION: Human β2m gene promoter is active in mouse mastocytoma P815 cells. It can regulate the expression of reporter gene under the control of IFN-γ.更多还原