绵羊高硫角蛋白启动子表达活性研究  

Study on the Activity of Sheep High-sulfur Keratin Promoter

作　　者：罗芳[1] 王春生[1] 刘欣[1] 安铁洙[1] 

机构地区：[1]东北林业大学生命科学学院,哈尔滨150040

出　　处：《中国畜牧兽医》2009年第6期61-64,共4页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(30771538);东北林业大学引进人才科研启动基金

摘　　要：角蛋白是羊毛主要结构蛋白,为了筛选在绵羊毛囊中具有高表达活性高硫角蛋白的内源启动子,本研究以绵羊基因组为模板,克隆得到高硫角蛋白基因启动子B2C,将B2C与去除CMV启动子的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传代培养的绵羊成纤维细胞和去除透明带的小鼠4细胞期胚,分析其表达活性。结果表明,转染后48 h,在蓝光激发条件下可以检测到绵羊成纤维细胞核内及核周围绿色荧光蛋白的高表达,转染72 h后,在核内表达减弱,而细胞质内表达增加;此外,虽然利用病毒启动子构建的GFP表达载体能够在小鼠的早期胚胎细胞中表达,但由B2C启动子与GFP构建的表达载体转染小鼠早期胚胎后,未观察到绿色荧光,表明B2C启动子在绵羊成纤维细胞中具有表达活性,但不能在小鼠早期胚胎中表达。The keratin is main structure albumen of the wool, which is the most important part of skin and hair. In order to detect the activity of high-sulfur keratin in sheep hair follicle, our experiment regarded the genomic DNA from the sheep as the template, through molecular biologic methods to clone the sheep high-sulfur keratin promoter B2C, then inserted it into pAcG- FP1-N1 plasmid which had been excised the CMV promoter. After PCR identifying, eukaryotic expression vector was con- structed. Sheep fibroblast and zona pellucida removal mouse embryos（4 cells） were transfected with the vector by cationic liposome method. After 48 hours transfection, under the excitation of blue light, green fluorescence could he seen nearby the nu- cleolus first. As time went, the fluorescence of nucleolus became dim, and the fluorescence in the cytoplasm became brighter gradually. After mouse embryos were transfected with the vector which bad virus promoter CMV, GFP could be found in mouse embryos, but GFP could be seen in mouse embryos transfected with the vector which had virus promoter B2C. The re- suits indicated that sheep high-sulfur keratin promoter B2C could start GFP expression in sheep fibroblasts and inactive in mouse embryos.

关 键 词：高硫角蛋白 启动子 分子克隆 活性分析 

分 类 号：Q78[生物学—分子生物学]