摘要
目的克隆肿瘤特异Ki-67启动子，观察其在人肿瘤细胞中的转录活性。方法提取。肾癌Ketr-3细胞基因组总DNA作为模板，聚合酶链反应（PCR）获取1．6kb的Ki-67启动子DNA片段。克隆到pGL3-Basic载体构建双荧光素酶检测质粒pGLB-Ki-67。将pGLB—Ki-67转染4种人肿瘤细胞及人脐静脉内皮Huvec细胞，使用双荧光素酶检测系统鉴定启动子活性及肿瘤特异性。结果电泳与测序结果显示克隆出的Ki-67启动子片段序列与Genbank中记录一致，pGLB．Ki-67质粒构建成功。其启动子活性在Hela、BIU-87、Ketr-3、A5494种人肿瘤细胞内分别达到SV40 promoter／enhancer活性的21．0％、31．1％、23．9％和7．2％；在Huvec细胞内仅为0．5％。结论克隆出Ki-67基因启动子（-820～＋771），并证明其具有良好转录活性。
Objective To clone DNA sequence of Ki-67 promoter,and identify its transcriptional activities in different human carcinoma cell lines. Methods The DNA fragment of Ki-67 promoter was cloned from the genomie DNA of Ketr-3 cells and inserted into lueiferase reporter vectors （pGL3-Basic） to reconstruct a recombinant plasmid named pGLB-Ki-67. The dual-lnciferase reporter assay system was used to identify the transcriptional activity and tumor specificity of Ki-67 in normal umbilical vein epithelial cell line Huvec and 4 different carcinoma cell lines （ Hela, BIU-87, Ketr-3 and A549 ）. Results Eleetrophoresis and DNA sequencing demonstrated that cloned Ki-67 promoter was 1.6 kb, as registered in Genbank. The recombinant plasmid pGLB-Ki-67 was comfirmed by DNA sequencing. In ela, BIU-87, Kerr-3 and A549 cells,the Ki-67 promoter exhibited different transcriptional activities respectively equivalent to 21. 0% ,31.1% ,23.9% and 7.2% of SV40 promoter/enhancer. In normal Huvec cells,the transcriptional activity of Ki-67 was only 0.5%. Conclusion We first cloned the Ki-67 promoter, which had a strong transcriptional activity and tumor specificity.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第1期87-88,共2页
Chinese Journal of Experimental Surgery
基金
基金项目：国家自然科学基金资助项目（30873021）
