RNA干扰抑制RPA70基因质粒载体的构建与鉴定  

The construction and identification of RNA interference inhibition plasmid vector of RPA70

作　　者：顾玉明[1] 王洵[1] 裴冬生 郑俊年 

机构地区：[1]徐州医学院附属医院介入放射科,江苏徐州221002 [2]江苏省肿瘤生物治疗重点实验室,江苏徐州221002

出　　处：《徐州医学院学报》2012年第12期815-818,共4页Acta Academiae Medicinae Xuzhou

摘　　要：目的构建含复制蛋白A70（RPA70）基因的短发夹RNA（shorthairpinRNA，shRNA）表达质粒，诱导RNA干扰（RNAinterference，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。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 LipofectamineTM 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.

关 键 词：复制蛋白A70 重组质粒 短发夹RNA RNA干扰 食管肿瘤 

分 类 号：R73-33[医药卫生—肿瘤]