【摘要】 【目的】筛选高效的foxp3RNA干扰的靶点用于阻断CD4+CD25+Treg细胞的免疫抑制功能。【方法】使用oligoengine软件设计foxp3基因shRNA序列,化学合成法合成4条shRNA序列,构建含foxp3的靶序列的慢病毒载体;构建出表达FLAG融合蛋白和目的基因的工具细胞293T细胞用于筛选RNA干扰的有效靶点。【结果】成功包装了4条foxp3基因靶序列相应的慢病毒颗粒;成功构建了表达融合蛋白和目的基因的293T工具细胞;在工具细胞上筛选出了2条RNAi效率分别为91.3%和95.6%的有效靶点。【结论】使用FLAG融合蛋白和慢病毒载体能够筛选出Foxp3基因的RNAi最佳靶点。更多还原

【Abstract】 [Objective] To screen the high effective RNA intervening targets of foxp3 gene to block the immunosuppression of CD4+CD25+Tregs. [Method] Four shRNA sequences were designed by oligoengine software and synthesized by chemical method. Lentiviral vector that consists target sequences of foxp3 was constructed. The 293T tool cell line that express FLAG fusion protein and target gene was constructed to screen the effective targets for RNA interference. [Result] Lentiviral vector that contains foxp3 cDNA sequences was constructed successfully. The 293T tool cells that express FLAG fusion protein and foxp3 gene fragments were constructed successfully. Two effective targets for RNAi were screened in 293T tool cells, the blocking efficiency were 91.3% and 95.6 % respectively. [Conclusion] The optimal targets of Foxp3 gene for RNAi can be screened by FLAG fusion protein and lentivirus vector.更多还原