靶向EphB4基因的shRNA真核表达载体的构建与鉴定  

Construction and Identification of pH1-EphB4-shRNA Expression Vector

作　　者：张艳荣[1] 董万利[1] 胡锦[2] 惠国桢[3] 

机构地区：[1]苏州大学附属第一医院神经内科,江苏苏州215006 [2]苏州大学附属第一医院神经外科,江苏苏州215006 [3]上海交通大学附属第六人民医院神经外科,上海200233

出　　处：《苏州大学学报（医学版）》2008年第3期349-352,共4页Suzhou University Journal of Medical Science

基　　金：国家自然科学基金项目(30371459);上海科学技术发展基金资助项目(034047)

摘　　要：目的构建和鉴定靶向EphB4基因的短发夹RNA(shRNA)真核表达载体。方法体外合成一对互补并编码靶向EphB4基因的特异性shRNA序列的寡核苷酸,克隆到经Bbs I酶切线性化的psiRNA人H1启动子质粒载体中,经酶切和测序鉴定所构建的重组载体是否正确;采用脂质体转染的方法将构建的重组载体导入人恶性胶质瘤细胞系U251中,采用逆转录-聚合酶链反应(RT-PCR)检测转染细胞EphB4mRNA的表达变化。结果经酶切和测序证明,pH1-EphB4-shRNA序列正确;转染pH1-EphB4-shRNA载体后,U251细胞EphB4基因的电泳条带明显减弱,在磷酸甘油醛脱氢酶(GAPDH)参比下较空载体组下降60%。结论靶向EphB4基因的shRNA真核表达载体构建成功,转染U251细胞之后获得稳定表达,并可特异性沉默EphB4基因的表达,为进一步研究EphB4基因在恶性胶质瘤的发生发展中的作用提供了实验基础。Objective To construct short hairpin RNA （shRNA） eukaryotic expression vector, targeting gene EphB4 which may play an important role in glioma pathogenesis. Methods A specific DNA oligonucleotide targeting gene EphB4 on appropriate site was synthesized and inserted into Bbs I linearized psiRNA-H1 vector. The sequence of the plasmid was identified by DNA sequencer and restriction endonuclease digestion. The recombinant plasmid was transfected into the glioma cell lines U251 by lipofection. After transfected 48 hours, the inhibition of EphB4 mRNA expression was detected by RT-PCR. Results It was verified that the specific DNA oligonu- cleotide was cloned into the vector successfully, and the expression of EphB4 mRNA in U251 cells was significantly reduced by 60% after transfecting the recombinant plasmid, compared to the controls. Conclusion It indicates the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of EphB4 mRNA, and those provides the precondition for the further study of EphB4 in glioma pathogenesis

关 键 词：EPHB4 短发夹RNA 恶性胶质瘤 载体构建 

分 类 号：R739.41[医药卫生—肿瘤]