RNaseⅢ制备的小分子干扰RNA（esiRNA）文库构建及优势分析  

Construction and advantage of endoribonuclease Ⅲ -prepared siRNAs （esiRNA） library

作　　者：李陪[1] 董智雄[1] 朱长军[1] 刘海燕[2] 

机构地区：[1]天津师范大学生命科学学院天津市动植物抗性重点实验室,300387 [2]泰山医学院附属医院,271000

出　　处：《国际生物医学工程杂志》2013年第1期1-4,8,共5页International Journal of Biomedical Engineering

基　　金：国家自然科学基金面上项目（30971446）;2011年教育部“新世纪优秀人才支持计划”项目（NCET-11-1066）;天津市应用基础与前沿技术研究计划重点项目（12JC2DJC21400）;天津师范大学中青年教授学术创新推进计划项目（52XC1001）;〈天津师范大学校级项目〉校博士基金（52XB1104）;天津师范大学“渤海学者”基金项目;泰山医学院科研计划课题项目（201021149）

摘　　要：目的建立KIF4A核糖核酸内切酶Ⅲ酶切制备的小分子干扰RNA（esiRNA）文库，并探讨这种新型siRNA制备方法的优势。方法制备502bp的KIF4A基因组序列作为转录模板；体外转录获得双链RNA；用核糖核酸内切酶ⅢGST融合蛋白（GST-RNaseⅢ）酶切双链RNA，制备KIF4AesiRNA文库；应用KIF4AesiRNA和化学合成的KIF4AsiRNA转染胃癌细胞株SGC-7901，应用Q-RTPCR和WesternBlot分别检测KIF4A的mRNA和蛋白水平。结果利用GST-RNaseⅢ成功制备了KIF4AesiRNA文库，该文库可有效抑制SGC．7901细胞内的KIF4A表达，且抑制效果明显优于化学合成的KIF4AsiRNA。结论用生物学方法制备的KIF4AesiRNA文库可有效抑制KIF4A在细胞内的表达，且抑制效果优于化学合成的siRNA。Objective To establish KIF4A 3＇UTR esiRNA library and analyze the advantage of method in studying the function of KIF4A in SGC-7901 cells. Methods The GST-fusion protein of Escherichia coli endoribonuclease Ⅲ （GST-RNase Ill ） was used to digest the double strand RNA （dsRNA）, which was transcribed in vitro from a 502bp template of KIF4A genome （T7-KIF4A 3＇UTR ）. The KIF4A esiRNA library generated from the above method and chemically synthesized KIF4A siRNA were then used to transfect SGC-7901 cells at 10 nmol/L and 20 nmol/L. Real-time quantitative PCR and Western Blot were used to detect the mRNA and protein level of KIF4A, respectively. Results The KIF4A esiRNA library was effectively established from dsRNA digestion using GST- RNase Ill. KIF4A expression was significantly reduced in SGC-7901 cells transfected with KIF4A esiRNA or siRNA. In addition, the inhibitory effect of KIF4A esiRNA was more effective than that of chemically synthesized siRNA. Conclusion KIF4A esiRNA library which was obtained using biological method can more effectively inhibited the expression of KIF4A more effectively in 5GC-7901 cells than chemically synthesized siRNA. Therefore, esiRNA library can be used as a new and more effective method in studying the function of KIF4A in SGC-7901 cells.

关 键 词：RNA干扰 esiRNA文库 KIF4A 

分 类 号：Q[生物学]