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目的对NGAL(neutrophilgelatinase-associatedlipocalin)基因5′侧翼区的转录调控启动子区进行分段定位鉴定。方法将NGAL基因5’侧翼区-416~+84和-152~+84两个区段分别克隆至专门用于研究启动子的报告基因表达载体pGL3-Enhancer(pGLE)中,构建表达载体pGLE-416和pGLE-152;然后将pGLE-152和pGLE-416分别与pRL-TK载体共转染HeLa细胞、EC109细胞和Vero细胞,通过检测相对荧光素酶活力,确认这些NGAL基因片段中是否含有启动子元件。结果与pGLE相比,pGLE-152在上述三种细胞中的相对荧光强度均明显增强(P<0.05),且在不同的细胞中增强的幅度明显不同。结论NGAL基因的启动子位于-152~+84区段内,启动子的强弱具有细胞特异性,这可能与增强子的协同作用相关联。
Objective To identify and identify the transcriptional regulatory promoter region of 5 ’flanking region of neutrophil gelatinase-associated lipocalin (NGAL) gene. Methods The two segments of -416 ~ +84 and -152 ~ + 84 of 5 ’flanking region of NGAL gene were cloned into pGL3-Enhancer (pGLE), which was used to study the promoter of promoter. The expression vector pGLE- 416 and pGLE-152. Then, pGLE-152 and pGLE-416 were co-transfected with pRL-TK vector respectively into HeLa cells, EC109 cells and Vero cells. The relative luciferase activity was tested to confirm whether these NGAL gene fragments contain promoter Sub-components. Results Compared with pGLE, the relative fluorescence intensity of pGLE-152 in all the three cells was significantly increased (P <0.05), and the amplitudes of pGLE-152 in different cells were significantly different. CONCLUSION: The promoter of NGAL gene is located in the -152 ~ +84 region. The promoter specificity is cell-specific, which may be related to the synergistic effect of the enhancer.