关键词: 牛Nramp1基因, 启动子活性, 调控区, LPS诱导

Abstract:

【Objective】Bovine natural resistance associated macrophage protein 1 gene (Nramp1) is a major anti-disease candidate gene, but its mechanism of transcriptional regulation remains unclear. To discover the Nramp1 gene expressional mechanism, its promoter region was determined, and the core sequences and major domains were found.【Method】 The 5′flanking region and different fixed 3′ terminal fragments of bovine Nramp1 were cloned and recombined into EGFP-N1 and/or pGL3-basic plasmids. After DNA sequencing confirmation, the recombined vectors were transfected into 293T and murine mammary epithelial cells RAW264.7, respectively, and treated with lipopolysaccharide (LPS), and then the promoter activities of different fragments of 5′flanking region of Nramp1 gene were determined by qualitative and quantitative assays including semi-quantitative RT-PCR and luciferase reporter gene analysis assay. 【Result】 The studies showed that the long DNA fragment, sequence from +58 to -89, and sequence up to -1 748 of 5′flanking region, had the stronger, basal, or maximal promoter activities, respectively. Further studies showed that there were positive (-89/-205 and -278/-1 495) and negative (-205/-278) regulatory domains, respectively. In addition, Escherichia coli LPS significantly enhanced the activity of Nramp1 promoter, and induced its expression in a dose-dependent and cell specific way. 【Conclusion】 The recombinant reporter plasmids containing different deduced fragments of Nramp1 promoter were successfully constructed, and its core and major regulatory regions were found.

Key words: bovine Nramp1 gene, promoter active, regulatory region, LPS inducing