论文部分内容阅读
目的构建表达靶向抑制survivin基因的三个短发夹结构(shRNA)的RNA干扰载体,使其在胶质瘤细胞发挥干扰效应。方法在survivin全长序列中选取设计3条19个核苷酸靶序列,2条反向重复序列,间以9个核苷酸的茎环序列,加上对应酶切位点,形成3条shRNA的DNA模板,分别克隆到3个干扰载体pG1,pG2和pG3上：采用分布酶切连接的方法,分别将U6启动子以及下游的后2个shRNA模板酶切下来,依次连接到pG1对应酶切位点,构建出含有3条shRNA模板且能独立编码shRNA的重组干扰载体pGenesil-survivin,测序鉴定：将干扰质粒导入到胶质瘤细胞U251,分别采用RT-PCR以及Western Bloting从mRNA和蛋白水平检测干扰后效果。结果重组的干扰载体含有3条正确的shRNA模板；RT-PCR以及Western Blotting检测显示,survivin的mRNA转录水平以及蛋白水平的表达均得到显著抑制。结论编码3条shRNA的干扰载体pGenesil-survivin介导的RNA干扰技术(RNAi)可以显著的靶向抑制survivin基因在人胶质瘤细胞U251中的表达。
OBJECTIVE: To construct RNA interference vectors expressing three short hairpin RNAs (shRNAs) targeting survivin gene and make them interfere with glioma cells. Methods Three 19 nucleotide target sequences and two inverted repeat sequences were designed and synthesized in the full-length sequence of survivin. Nine stem-loop sequences of 9 nucleotides were added together with corresponding restriction sites to form 3 shRNAs Were cloned into three interference vectors, pG1, pG2 and pG3, respectively: The U6 promoter and the downstream two shRNA templates were digested with restriction enzyme digestion and ligated into pG1 corresponding digestion The recombinant plasmids pGenesil-survivin containing 3 shRNA templates were constructed and sequenced. The plasmids were transfected into glioma U251 cells by RT-PCR and Western Bloting respectively. From the mRNA and protein levels After detecting the effect of interference. Results The recombinant plasmid contained three correct shRNA templates. The mRNA and protein levels of survivin were significantly inhibited by RT-PCR and Western Blotting. CONCLUSION: RNA interference (RNAi) mediated by pGenesil-survivin, a three-shRNA shRNA, can significantly inhibit the expression of survivin gene in human glioma U251 cells.