中文摘要：

以绵羊基因组DNA为模板克隆获得绵羊角蛋白结合蛋白基因KAP6.1启动子。将KAP6.1启动子插入到切除了CMV启动子的pAcGFP1-N1质粒中,构建了重组真核表达载体,并采用脂质体法转染传代培养绵羊皮肤成纤维细胞并检测其表达活性。结果显示：（1）KAP6.1启动子序列大小为830 bp,与GenBank上绵羊角蛋白结合蛋白的参照序列（M95719）有99.64%的同源性;与M95719相比,KAP6.1在349位点、726位点和246位点上分别存在1个C缺失、1个转换（G代替A）和1个颠换（A代替T）;在438位点上具有1个依赖DNA的RNA聚合酶结合位点TATA box,但未发现具有依赖DNA的RNA聚合酶识别位点CAAT box;（2）转染24 h后,在蓝光激发下检测到细胞核附近有绿色荧光蛋白的高表达,并在48 h后逐渐减弱,而细胞质内绿色荧光蛋白的表达量增加。这一结果表明,KAP6.1启动子在绵羊皮肤成纤维细胞的表达上可能具有特异性。

英文摘要：

To clone the promoter which expresses specifically in hair follicle and examine its activity in fibroblasts are necessary for construction of the specific expressing vector in hair follicle.Therefore,this experiment regarded the genomic DNA from the sheep as the template to amplify KAP6.1 promoter by PCR.Sequencing analysis showed that fragment of KAP6.1 contained 830 nucleotides and shared a homology of 99.64% with the sequence（M95719） on GenBank.There were 3 differences between the promoter and the reported sequence： at the region of 349 bp,C was flawed;at the region of 438 bp,A was changed by G;and at the region of 726 bp,T was changed by A.There is a TATA box that depended on DNA at 246 bp,whereas no CAAT box was found in the DNA fragment.Then the expression vector in eukaryotic cells was constructed successfully by replacing CMV promoter with KAP6.1 in pAcGFP1-N1 vector.At last,the expression vector was transfected into ovine fibroblast through cationic liposome method.After trsanfected into the sheep fibroblasts for 24 hour,green fluorescence occurred nearby the nucleolus first.As time went,the fluorescence of nucleolus became dim,and the fluorescence in the cytoplasm became brighter gradually.