高级搜索

NGAL 基因5'侧翼区启动子区的克隆与鉴定

Cloning and Identification of 5'Flanking Region of Promoter in NGAL Gene

  • 摘要: 目的 对NGAL(neutrophil gelatimse-associated lipocalin)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。

     

    Abstract: Objective  Cloning and identifing of 5'flanking region promoter in NGAL ( neut rophil gelatinase - associated lipocalin) gene. Methods  Two different length f ragments -416~+ 84 and -152~+84 of 5'flanking region of NGAL gene were cloned into pGL3-Enhancer vector (pGLE), which aids in verification of functional promoter element s. And pGLE-416 and pGLE-152 reporter gene vectors were const ructed. Then they were respectively cot ransfected with pRL-TK vector into HeLa, EC109 and Vero cells. Relative light unit (RLU) in the cells was measured with Dual Luciferase Report Gene Assay System (DLR) to prove whether there are promoter element s in these f ragments. Results  RLU of pGLE-152 was obviously increased (P<0. 05), compared with control pGLE, after they had been transfected into three different cell lines. Moreover, there was variable fold induction in the different cells. Conclusion  The promoter of NGAL gene is located in-152~+84 region and has a cellular specialty, which might relate to cooperation with enhancer elements.

     

/

返回文章
返回