【摘要】 目的 构建含复制蛋白A 70(RPA70)基因的短发夹RNA(short hairpin RNA,shRNA)表达质粒,诱导RNA干扰(RNA interference,RNAi)。方法 根据GenBank中RPA70基因的mRNA序列,设计、合成2条反向重复多聚寡核苷酸序列,退火形成双链DNA。利用分子克隆技术,将含RPA70基因的双链DNA与经双酶切后的载体pSi-U6-GFP-Neo连接,构建pSi-U6-GFP-Neo-shRNA-RPA70重组质粒,通过DNA测序证实表达质粒构建成功。设空白对照组、阴性对照组及实验组,在脂质体2000的介导下转染食管癌TE-1细胞株,荧光显微镜下观察绿色荧光蛋白(GFP)的表达情况。结果 DNA测序证实含RPA70基因的shRNA表达质粒构建成功。结论 成功地构建了重组质粒pSi-U6-GFP-Neo-shRNA-RPA70,为下一步干扰实验奠定了基础。更多还原

【Abstract】 Objective To construct recombinant plasmids generating short hairpin RNA（shRNA） in mammalian cells so as to induce RNA interference（RNAi）.Methods Two oligonucleotides for replication protein A 70（RPA70）mRNA of GenBank were designed,double-stranded DNA which dropped temperature.Then using cloning technique,double-stranded DNA which contained RPA70 was cloned into pSi-U6-GFP-Neo vector digested by two restricted endoenzymes according to its special orientation.Three groups were studied:blank control group,negative control and antisense transfected group.Human esophageal carcinoma cell line TE-1 was transfected with recombinant plasmid by Lipofectamine^TM 2000.Results DNA sequencing showed that the sequence of recombinant vector pSi-U6-GFP-Neo-shRNA-RPA70 was successfully constructed.Conclusion The recombinant vector pSi-U6-GFP-Neo-shRNA-RPA70 was sueeessfully constructed,which provides foundation for the RNAi experiment.更多还原