摘要
目的:构建并筛选肿瘤坏死因子受体相关因子6(TRAF6)短发夹RNA(shRNA)表达质粒.方法:针对小鼠TRAF6 mRNA设计4条理论上最佳的siRNA序列,经退火成互补双链,将相应双链DNA插入pGCsi-U6/GFP/Hygro质粒中,构建重组表达质粒pGCsi-TRAF6-shRNA1,2,3,4,并以不同比例DNA质粒/脂质体转染重组质粒(1∶2、2∶5、1∶3和1∶4)至RAW264.7细胞中,观察转染效果.结果:靶向TRAF6 mRNA的4个shRNA重组质粒载体pGCsi-TRAF6 shRNA1,2,3,4,经测序分析,shRNA编码序列与设计的片段完全一致,证实载体构建成功.应用荧光显微镜分析转染效率显示,DNA(g)/脂质体转染重组质粒(L)按1∶2、2∶5、1∶3和1∶4比例转染细胞的效率分别为13.7%±1.2%、24.5%±2.1%、19.3%±1.7%、16.3%±2.8%,以2∶5为最佳比例.只加了脂质体未加质粒(试剂对照)的细胞无荧光表达.结论:TRAF6靶向RNA干扰重组表达质粒构建成功,为进一步研究阻断TRAF6表达对急性肝衰竭过度炎症反应的基因治疗奠定基础.
AIM： To construct and select TNF receptorassociated factor 6 （TRAF6） short hairpin RNA （shRNA） expressing plasmid that can inhibit TRAF6 mRNA expression in Raw 264.7 cells. METHODS： Four pairs of oligos for hairpin RNA targeting mouse TRAF6 gene were chemically synthesized. The annealed oligos were inserted into the down stream of U6 promoter of linearized pGCsi-U6/GFP/Hygro vector to construct RNA interference （RNAi） plasmid （pGCsiTRAF6 shRNA） respectively. To get most effective and optimal dosage siRNA, the four vectors were transfected into Raw 264.7 cells with differ- ent ratios between plasmid （g） and TransFectin （L） （1 ： 2, 2 ： 5, 1 ： 3 and I ： 4）, and the expression of fluorescence and efficiency of transfection were detected by fluorescence microscopy. RESULTS： The recombinant plasmids pGCsi- TRAF6 shRNA1, 2, 3, 4 were successfully constructed and the inserted sequence was confirmed by DNA sequencing. Fluorescence microscopy showed that the transfection efficiencies were 13.7% ± 1.2%, 24.5% ＋ 2.1%, 19.3% ±1.7% and 16.3% ±2.8% at ratios of 1 ： 2, 2 ： 5, 1 ： 3 and 1 ： 4, respectively, between plasmid （g） and TransFectin. The ratio of 2 ： 5 was considered as the optimal one. CONCLUSION： The siRNA plasmid targeting mouse TRAF6 gene is successfully developed and can be applied to study the function of TRAF6 on inflammatory reaction during acute hepatic failure （AHF）.
出处
《世界华人消化杂志》
CAS
北大核心
2009年第14期1406-1411,共6页
World Chinese Journal of Digestology
关键词
肿瘤坏死因子受体相关因子6
RNA干扰
重组表达质粒
短发卡RNA
Tumour necrosis factor receptor-associ-ated factor 6
RNA interference
Eukaryotic expres-sion vector
Short hairpin RNA
