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.
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)
