Y-盒结合蛋白1短发夹环RNA真核表达载体的构建  

Generation of eukaryotic expression vector of shRNA specific for Y-box binding protein-1

作　　者：周磊磊[1] 许文林[1] 秦茹娟[1] 唐华容[1] 沈慧玲[2] 

机构地区：[1]江苏大学附属人民医院中心实验室,江苏镇江212002 [2]江苏大学附属人民医院肿瘤科,江苏镇江212002

出　　处：《江苏大学学报（医学版）》2007年第1期35-37,65,共4页Journal of Jiangsu University：Medicine Edition

基　　金：江苏省卫生重大课题基金资助项目(K2005017)

摘　　要：目的:构建Y-盒结合蛋白1(Y-box binding protein-1,YB-1)特异性短发夹环RNA(shRNA)真核表达载体。方法:采用人U6SnRNA启动子,分别把被9 bp序列间隔的19 bp长短的YB-1靶序列的反向重复序列,置于pGensil-1质粒载体中,构建成YB-1短发夹环RNA,产生重组质粒pGensil-1/U6/YBX11,pGensil-1/U6/YBX12及pGensil-1/U6/YBX13。分别用PstⅠ和SalⅠ酶切鉴定;并将经酶切鉴定正确后的重组质粒作测序分析。结果:YB-1的特异性shR-NA片段被成功克隆进pGensil-1质粒中,SalⅠ酶切鉴定可切出400 bp大小的片段,DNA测序分析显示,重组质粒shRNA编码序列与设计片断的序列完全一致。结论:成功构建了针对YB-1的特异性shRNA真核表达载体pGensil-1/U6/YBX11,pGensil-1/U6/YBX12及pGensil-1/U6/YBX13,为进一步研究其基因功能打下了基础。Objective： To generate eukaryotic expression vector of short hairpin RNA （shRNA） specific for Y-box binding protein-1 （YB-1）. Methods： A plasmid pGensil-1 containing human U6SnRNA promoter was legated to a 19 bp reverse repeated motif of YB-1 target sequence with 9 bp spacer. Enzyme digestion and DNA sequencing were used to examine whether the recombinant plasmid pGensil-1/U6/YBX11, pGen- sil-1/U6/YBX12 or pGensil-1/U6/YBX13 was correct. Results： The YB-1 shRNA expression frame was successfully inserted into eukaryotic expression vector pGensil-1 with U6SnRNA promoter and termination signal of RNA polymerase Ⅲ. Enzyme digestion by Pst I and Sal I got a 400bp DNA fragment. The sequences of shRNA recombinant plasmid were the same as that of designed fragments. Conclusion： Eukaryotic expression vectors of shRNA specific for YB-1 were generated successfully. Our study may start a new approach to research the gene function of YB-1 and the method of gene therapy.

关 键 词：RNA干扰 YB-1 短发夹环RNA U6SnRNA启动子 

分 类 号：R392.1[医药卫生—免疫学]