人类生存蛋白启动子的克隆及其在淋巴瘤细胞中的转录活性研究  

Clone of the Survivin promoter and its specific transcriptional activity in human lymphoma cells

作　　者：唐友红[1] 钟美佐[1] 

机构地区：[1]中南大学湘雅医院肿瘤科,长沙410008

出　　处：《白血病．淋巴瘤》2010年第2期84-87,共4页Journal of Leukemia & Lymphoma

基　　金：教育部博士点基金（20090162110045）

摘　　要：目的克隆人类生存蛋白（Survivin）核心启动子，研究Survivin启动子在人类淋巴瘤细胞Ramos和健康人肝脏细胞ChangLiver中的转录活性。方法以人类肠基因组DNA为模板，PCR扩增Survivin启动子987bp片段，将其通过酶切位点连人pGL3-Basic载体构建pGL3-Survivin荧光素酶报告基因载体，通过脂质体转染法转染Ramos和ChangLiver细胞，通过检测荧光素酶表达水平，比较Survivin启动子在这两种细胞中的转录活性。结果成功克隆出987bp的Survivin启动子；双酶切、PCR检测和DNA测序证实pGL3-Survivin载体构建成功；荧光素酶活性检查显示：Survivin启动子在Ramos细胞中的转录活性为阳性对照CMV启动子活性的4．5％，明显高于在ChangLiver细胞中的0．19％，在淋巴瘤细胞中具有较高特异性，且在淋巴瘤细胞中的活性明显高于阴性对照pGL3-Basic的0．086％。结论Survivin启动子在淋巴瘤细胞中具有较高的特异性，可以作为淋巴瘤细胞基因转染的肿瘤特异性启动子使用。Objective To clone core promoter of human Survivin gene, and investigate the transcriptional activity of the Survivin promoter in the human lymphoma cells Ramos and the normal human liver cells Chang Liver. Methods The Survivin promoter was amplified by PCR using the total genomic DNA of human colon as sample DNA and then cloned into pGL3-Basic vector. The reporter gene vector was named pGL3-Survivin. This vector was transfected into Ramos cells and the control Chang Liver cells to detect the transcriptional activities of Survivn promoter by cationic liposome. Results The Survivin promoter was amplified through PCR successfully. The results of restriction enzyme digestion, PCR and sequencing indicated that the pGL3-Survivin luciferase expression vector was constructed triumphantly. Survivin promoter showed much more higher transcriptional activity in Ramos cells than in Chang Liver cells. Relatively, pGL3- CMV and pGL3-Basic separately showed no obviously different transcriptional activity in Ramos cells and Chang Liver cells. Conslusion The Survivn promoter has lymphoma specificity and may be used as tumor specific promoter in gene transfection of lymphoma.

关 键 词：启动区(遗传子) 转录启动子 基因载体 荧光素酶 淋巴瘤 

分 类 号：R733.1[医药卫生—肿瘤]