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

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。

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

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 oligonucleotides. The result of RT-PCR showed that Rab25 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.