针对DEK原癌基因的siRNA真核表达载体的构建  

Construction of siRNA vector accorded to DEK oncogene

作　　者：刘岿然[1] 杨雨[1] 冯天达[2] 张淑兰[1] 

机构地区：[1]中国医科大学盛京医院妇产科,辽宁沈阳110004 [2]中国医科大学盛京医院神经外科,辽宁沈阳110004

出　　处：《中国现代医学杂志》2010年第20期3053-3056,共4页China Journal of Modern Medicine

基　　金：辽宁省高等学校科研项目计划(No:2008862)

摘　　要：目的以 DEK基因为靶基因,针对其特定核苷酸序列,构建psiRNA-hHDEK表达载体,转染宫颈癌CaSki细胞,观察DEK siRNA对DEK基因mRNA转录水平和蛋白表达水平的靶向沉默效应。方法根据DEK核苷酸序列,利用软件设计出针对DEK位点的特异性小干扰RNA,克隆至siRNA真核表达载体psiR-NA-hHl neo中,构建能在真核细胞中表达的靶向DEK基因的siRNA重组质粒;应用脂质体介导重组质粒转染宫颈癌CaSki细胞株,用RT-PCR和Western Blot检测转染后DEK mRNA和蛋白表达。结果筛选出阳性克隆,抽提质粒,经限制性内切酶AseI酶切鉴定及测序分析证实合成的siRNA序列正确,并已准确克隆入psiRNA-hHl neo载体中。DEK特异性siRNA载体构建成功。经RT-PCR和Western Blot测定分析,转染质粒psiRNA-hHDEK CaSki细胞组DEK mRNA和蛋白的表达明显减少。结论成功构建了DEK特异性siRNA表达载体。设计构建的真核表达载体可以特异性干扰DEK原癌基因的表达,为进一步研究DEK基因的功能奠定了基础。Objective DEK target siRNA was designed according to its specific nucleotide sequence, and cloned into psiRNA hHDEK expression vector to construct a recombinant plasmid. It was transfected into CaSki cervical cancer by lipofectamine. Its expression at mRNA and protein level were examined by RT-PCR and western blot to inquire the relationship between DEK and cervical cancer. MethodsAccording to nucleotide sequence in Genebank. The recombinant DEK siRNA plasmid was evaluated by enzyme digestion and sequencing. Then it was transfected into CaSki cells by lipofectamine. DEK mRNA and protein expression were examined to verify the gene silence effect at mRNA transcription and protein expression level by RTPCR and Western blot analysis. Results more than 10 positive clones were screened in the kanamycin plates, plasmid was extracted, identified by AseI endonuclease digestion, it suggested that specific DEK siRNA vector was constructed successfully. DEK mRNA and protein expression was significantly reduced in psiRNA-hHDEK transfected CaSki cells, lower than that of psiRNA-hH1neo transfected group and the non-transfected group. Conclusions DEK siRNA eukaryotic expression vector was constructed successfully and made DEK gene expression of CaSki cells silence effectively. The specific DEK siRNA vector could inhibit the expression of DEK gene, which forms the basis for investigating DEK gene＇s function.

关 键 词：宫颈癌 DEK RNA干扰 转染 

分 类 号：R711.74[医药卫生—妇产科学] R737.33[医药卫生—临床医学]