线粒体细胞色素C氧化酶RNAi慢病毒载体的构建-中国科技论文在线.PDFVIP

中国病理生理杂志 Chinese Journal of Pathophysiology 2005，21(6)：1117—1123 · lll7 · [文章编号]1000—4718(2005)06—1117—07 线粒体细胞色素C氧化酶RNAi慢病毒载体的构建* 陈 艳 ， 邵建永 ， 吴秋良 ， 江高峰 ， 夏云飞 ， 陈忠平 (中山大学肿瘤防治中心 病理科，实验研究部，广东广州510060) [摘 要] 目的：通过构建携带细胞色素C氧化酶基因的RNAi慢病毒载体，获得可供转染的滴度，为下一步研 究该基因缺陷在真核细胞中的影响提供物质基础。方法：根据线粒体细胞色素C氧化酶设计的两条互补的单链寡 核苷酸退火后形成双链，插入到pENTR／U6质粒缺口末端，连接在质粒上生成含RNAi盒的pENTR／U6载体；通过重 组作用将pENTR／U6载体的RNAi盒重组到pLenti6／BLOCK—iT—Dest载体上，构建含U6启动子、靶序列和P0lⅢ终止 子表达框的MTCOX—I shRNA表达重组体；经脂质体导入293FT细胞，包装成慢病毒，收集病毒上清并检测其滴度。 Western blotting检测干扰后细胞内线粒体细胞色素C氧化酶I亚基的表达。结果：将目的序列成功连接到载体上，并 经测序分析证实载体构建成功；成功包装成高滴度的慢病毒。Western blotting检测结果证实构建的MTCOX—I shRNA 表达重组体可显著抑制线粒体细胞色素C氧化酶I亚基的表达。结论：成功构建了携带细胞色素C氧化酶基因的 RNAi慢病毒载体。 [关键词]基因；线粒体；重组；慢病毒属；载体；细胞色素C氧化酶 [中图分类号]R730．54；Q785 [文献标识码] A The construction of lentivirus——mediated RNAi vector containing cytochrome C oxidase C}玎 N Yah ，SHAO Jian—yong ，WU Qiu—liang ，JIANG Gao—feng2，XIA Yun—fei ， CHEN Zhong—ping (‘Department ofPathology， Department ofResearch，Cancer Center，Sun Yat—sett University，Guangzhou 510060，Ch／na) [AB!汀RACT] A卫M：To constrict a recombinant lentivirus RNAi vector can-ying cytochrome C oxidase gene to obtain the titer ofthe lentiviral stock for investigation ofthe expression in the eukaryotic cell and the affection ofthe COX gene silencing in the eukaryotic cells．METHODS：According to the DNA of the cytochrome C oxidase gene，we designed and synthesized complemen— tary single—strand DNA oligos，annealed the single—stranded oligos to generate a ds oligo．cloned the ds oligo into pENTR／U6 to obtain an entry clone；An LR recomb ination reaction was performed between the pENTR／U6 entry construct and pLenti6／BLOCK— iT—Dest to generate expression construct，the 293FT cell line Was co