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目的:将不同长度的NGAL基因 3′端序列(包括外显子、内含子和部分3′侧 翼非翻译序列)克隆到pGL3 Promote (pGLP)载体中,通过检测报告基因荧光素 酶活力,确认NGAL3′端序列中是否含有增 强子或抑制子元件。方法:应用PCR技术 从SHEEC细胞基因组DNA中扩增出不同 长度的NGAL基因片段F5(1880bp)和F (826bp),将其克隆到pGEM Teasy载体 并测序验证;再亚克隆到pGL3 Promoter载 体中,获得pGLP F5和pGLP F7表达载体 将pGLP F5和pGLP F7载体分别与pRL TK载体共转染HeLa、EC109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增强子元件 结果:我们成功构建了pGLP F5和pGLP F 重组载体。Hela、EC109和Vero转染细胞 与pGL3 Promoter相比,酶活力未表现出明 显的增强或减弱。结论:在本研究的实验 条件下,NGAL基因F5和F7片段内潜在的 顺式作用元件并没有发挥作用,或作用不 明显。
OBJECTIVE: To clone the 3 ’end of NGAL gene of different length (including exon, intron and partial 3’ untranslated sequence) into pGL3 Promote (pGLP) vector and detect the reporter luciferase activity and confirm NGAL3 ’end sequences contain enhancer or repressor elements. METHODS: F5 (1880bp) and F (826bp) of NGAL gene fragments of different length were amplified by PCR from genomic DNA of SHEEC cells. The fragments were cloned into pGEM Teasy vector and verified by sequencing. Subcloned into pGL3 Promoter vector, Obtaining pGLP F5 and pGLP F7 expression vectors The pGLP F5 and pGLP F7 vectors were cotransfected with pRL TK vector respectively into HeLa cells. EC109 and Vero cells were tested for relative luciferase activity to confirm whether these NGAL gene fragments contain enhancer element results: We successfully constructed the pGLP F5 and pGLP F recombinant vectors. Hela, EC109 and Vero transfected cells showed no significant increase or decrease in enzyme activity compared to pGL3 Promoter. Conclusion: Under the experimental conditions in this study, potential cis-acting elements in F5 and F7 fragments of NGAL gene did not play a role, or the effect was not obvious.