Rab25基因siRNA表达载体的构建鉴定及在人卵巢癌细胞A2780中的表达  

Construction and identification of Rab25 siRNA expression vector and its expression in oarian carcinoma cell line A2780

作　　者：樊杨[1] 辛晓燕 陈必良 马向东 王珑[3] 商莉[1] 

机构地区：[1]宁夏自治区人民医院妇产科,宁夏银川750021 [2]第四军医大学第一附属医院妇产科,陕西西安710032 [3]宁夏医学院口腔系,宁夏银川750004

出　　处：《中国现代医学杂志》2007年第15期1822-1825,共4页China Journal of Modern Medicine

摘　　要：目的为研究Rab25基因的功能及卵巢癌的基因治疗,构建针对Rab25基因的siRNA表达载体,转染细胞A2780后观察其对Rab25基因表达的抑制作用,为探索卵巢癌基因治疗的新途径打好基础。方法根据基因库上的Rab25mRNA序列,设计并合成两端含有酶切位点的64个碱基的寡核苷酸链。寡核苷酸链退火后用T4DNA连接酶连接到线性化的pSUPER质粒中,并对重组质粒(命名为pSUPER/Rab25siRNA)进行酶切及序列鉴定,后转染卵巢癌细胞A2780,RT-PCR检测转染前后Rab25的表达情况。结果双酶切证实Rab25siRNA表达载体克隆构建成功,插入片段测序结果与合成的siRNA结果一致。RT-PCR检测显示转染卵巢癌细胞A2780后有效抑制了Rab25基因的表达。结论成功构建Rab25siRNA表达载体,为卵巢癌基因治疗开辟新途径。[Objective] To construct Rab25 siRNA expression vector in order to explore a new method of ovarian cancer gene therapy, and to assess its function on ovarian carcinoma cell A2780. [Methods] According to Rab25 mRNA sequence in the Genebank, a pair of 64-nt oligonucleotides, each containing the sites of restriction endonuclease at both ends, were designed and synthesized. Oligonucleotides were annealed and ligated with linearized pSUPER by T4DNA ligase. The recombinants （named pSUPER/Rab25 siRNA ） were finally sequenced and identified by enzyme cutting and sequencing. RT-PCR analysis was taken to show the change of Rab25 after the constructed plasmid had been transfeeted into A2780 cells. [Results] Rab25 siRNA expression vector was successfully constructed and identified by double endonuclease digestion. Sequence analysis of inserted fragment revealed the same sequence as synthesized siRNA oligonueleotides. The result of RT-PCR showed that Rub25 siRNA plasmid had inhibited Rab25 expression in A2780 cells obviously. [Conclusion] Rab25 siRNA expression vector has been successfully constructed, which will facilitate further studies of Rab25 function and its application in the treatment of ovarian cancer.

关 键 词：RAB25 SIRNA 载体构建 鉴定 卵巢癌 表达 

分 类 号：R737.31[医药卫生—肿瘤]