小鼠超高硫角蛋白基因启动子的克隆及其表达活性分析

Cloning and Activity of Mouse Ultra-High Sulfur Keratin Gene Promoter

目的筛选在小鼠毛囊中具有高表达活性的内源启动子,为建立小鼠被毛特异表达外源蛋白的转基因技术奠定基础。方法以小鼠基因组为模板,克隆得到超高硫角蛋白基因启动子超高硫角蛋白(UHS),将其分别与pβgal-Basic和pAcGFP1-N1载体连接,构建了重组真核表达载体。采用阳离子脂质体法转染胎鼠组织块,分析其表达活性。结果转染后48 h,在蓝光激发条件下可以检测到绿色荧光蛋白(GFP)在小鼠毛囊区高表达,转染96 h后,表达减弱;此外,转染后48 h,βgal染色结果显示在皮肤块的毛囊区存在蓝色点状区域。结论 UHS启动子在小鼠毛囊中具有表达特异性。

Objective Keratin is the main structure albumen of the mammal wool,which is the most important part of the skin,hair and nail,etc.The aim of this study was to screen ultra-high sulfur keratin promoter in the hair follicles of mouse skin,and provide a basis for establishment of a transgenic technique for hair-specific expression of exogeneous protein.Methods Regarding the genomic DNA from the mouse as a template,the mouse ultra-high sulfur keratin promoter（UHS） was cloned using molecular biologic methods,identified after conjugation with pMD18 simple vector and then insert it into pAcGFP-N1 plasmid,which had been excised the CMV promoter.After identification by PCR,a recombinant eukaryotic expression vector was constructed.At the same time,the promoter was linked to pβgal-basic,and recombination plasmid U-GFP and U-β-gal were obtained,respectively.The mouse skin tissue pieces were transfected by cationic liposomes of this plasmids.Results GFP expression was detected at mouse hair follicle region under blue light ilumination at 48 h after transfection,and the expression was decreased at 96 h after transfection.Moreover,at 48 h after transfection,blue dots of βgal staining were seen at the mouse hair follicle region.Conclusion The UHS promoter is a tissue-specific promoter in the mouse hair follicles.