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

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 Cloning and identifing of 5’flanking region promoter in NGAL (neutrophil gelatinase-associated lipocalin) gene. Methods Two different length fragments -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 elements. And pGLE -416 and pGLE -152 reporter gene vectors were constructed. Then they were respectively cotransfected 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 elements in these fragments. Results RLU of pGLE-152 was obviousmy 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.