真核生物表达成熟体miR-449a及其生物学活性的研究  

The Expressing of Mature miR-449 in Eukaryotes and the Investigation of Its Biological Activity

作　　者：武佳[1] 杨耿兵[1] 李伟[1] 娄文加[1] 马清华[1] 徐艳敏[1] 钱程[1] 刘立[1] 

机构地区：[1]浙江理工大学生命科学学院,杭州310018

出　　处：《浙江理工大学学报（自然科学版）》2010年第5期784-788,共5页Journal of Zhejiang Sci-Tech University(Natural Sciences)

基　　金：国家重点基础发展计划973(2004CB518804)

摘　　要：通过分子克隆技术将pre-miR-449a及其上下游200 bp序列克隆至pcDNA3.0多克隆位点,采用脂质体转染该载体至人宫颈癌Hela细胞中,Real-Time PCR检测载体的表达能力,同时使用荧光淬灭法检测表达产生的miRNA的生物学活性。结果表明:转染实验组载体的Hela细胞miR-449a的表达明显升高,而对照组表达几乎无变化,显示真核表达载体pcDNA3.0可以高效表达miRNA;此外,目的质粒与报告质粒共转染Hela细胞后绿色荧光明显淬灭而对照组荧光未淬灭,证明表达的miRNA具有生物学活性。To take advantage of the technology of molecular clone,the pre-miR-449a and it＇s flanking 200bp sequence is cloned to the multi-clone site（MCS） of pcDNA3.0,then the liposome carries the plasmid to transfect the human cervical carcinoma cell（Hela）,detecting the expression of miR-449a by Real-Time PCR.Then,the authors use fluorescence quench to determine the biologic activity of mature miRNA which expressed by pcDNA3.0-miR-449a.The data shows： the express level of experimental group of Hela is much more higher than control group.So,pcDNA3.0 can efficiently express mature miRNA which have biologic activity.

关 键 词：PCDNA3.0 miR-449a 人宫颈癌细胞 

分 类 号：R73-362[医药卫生—肿瘤]