Cyclin E基因siRNA真核载体的构建及干扰效果的初步鉴定  

Construction of siRNA Eukaryotic Vector of Cyclin E Gene and Preliminary Identification of Its Interference Efficiency

作　　者：陈济[1] 潘巍巍[1] 魏丽丽[1] 袁成福[1] 易发平[1] 卜友泉[1] 宋方洲[1] 

机构地区：[1]重庆医科大学生物化学与分子生物学教研室,临床检验诊断学省部共建教育部重点实验室,重庆400016

出　　处：《西南大学学报（自然科学版）》2008年第1期69-74,共6页Journal of Southwest University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(No:30371479)

摘　　要：根据CyclinE(细胞周期蛋白E)基因序列,设计siRNA干扰序列,将带9个碱基茎环结构的干扰序列插入带U6启动子真核表达载体,构建针对CyclinE基因的shRNA真核表达载体.酶切和测序鉴定确认载体构建成功后,脂质体法转染宫颈癌HeLa细胞,观察绿色荧光蛋白表达检测转染效率,RT-PCR法检测载体对CyclinE基因的表达抑制效果.实验结果显示CyclinE基因的RNAi真核表达载体构建成功,转入HeLa细胞后可以特异抑制细胞中CyclinE基因的表达.其中靶向(+797^+819)位点的质粒干扰效果较好,基因表达抑制率为59%.这些结果为利用该载体抑制CyclinE基因进行宫颈癌基因治疗研究奠定了基础.The gene sequence of Cyclin E was found in GenBank and the interference sequences were designed following siRNA design rules. A hairpin loop with 9 base pairs was added into the interference sequence and inserted into the eukaryotic vectors which contain the U6 promoter. The shRNA eukaryotic vectors special for knocking down Cyclin E gene were constructed. Verified by enzyme digestion and sequencing, the vectors were transfected into HeLa cells. The transfection efficiency was reported by green fluorescence protein expression and gene inhibition efficiency was analyzed by reverse transcription polymerase chain reaction （RT-PCR）. The result indicated the eukaryotic vectors special for knocking down Cyclin knock E gene were successfully constructed. Having been transfected into HeLa cell they can specially down the expression of Cyclin E gene. The plasmid aiming at the gene site（＋797～＋819）had the better inhibition effect （inhibition rate was 0.59）. The vector can be used in further study for treating cervical cancer by inhibiting the expression of Cyclin E gene.

关 键 词：RNA干扰 细胞周期蛋白E HELA细胞 细胞周期 

分 类 号：R737.33[医药卫生—肿瘤]