关键词: 高硫角蛋白; 启动子; 分子克隆; 活性分析

Abstract: 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 pAcGFP1-N1 plasmid which had been excised the CMV promoter. After PCR identifying, eukaryotic expression vector was constructed. 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 be seen nearby the nucleolus 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 had 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 results indicated that sheep high-sulfur keratin promoter B2C could start GFP expression in sheep fibroblasts and inactive in mouse embryos.

Key words: high-sulfur keratin; promoter; molecular cloning; activity analysis