TLR5干涉慢病毒颗粒制备及稳定细胞系建立

目的 制备Toll样受体5(TLR5)的慢病毒干涉颗粒,建立稳定下调MyD88的HEK293细胞系.方法 将携带不同特异性干涉序列的DNA片段插入干涉质粒pSicoR中,并制备慢病毒颗粒.将慢病毒颗粒感染HEK293细胞,建立稳定细胞系,通过RT-PCR和报告基因分析的方法确定不同干涉序列对TLR5表达的下调及信号通路启动的阻断作用.结果 成功制备携带干涉序列的慢病毒颗粒,并建立稳定感染慢病毒的HEK293细胞系.与对照组相比,感染2325位序列的细胞TLR5 mRNA表达水平下降为0.32,对CBLB502的启动作用下降为0.36.结论 成功制备TLR5的慢病毒干涉颗粒,并建立TLR5稳定下调的细胞系.

Packaging and identification of lentivirus particles interfering TLR5

Objectives To reduce expression of Toll-like receptor 5（TLR5）,lentivirus particles bearing various interfering sequences,and to construct HEK293 cell line consistently down-regulating TLR5.Methods Cloning DNA segments including specific interfering sequences were packaged into lentivirus particles.Stable cell lines were established after infecting HEK293 cell with lentivirus particles.Analysis of efficiency of RNA interference（RNAi） on TLR5 and signal pathway activations was detected by RT-PCR and reporter gene analysis.Results Lentivirus particles bearing various interfering sequences were packaged and stable HEK293 cell lines infected by lentivirus were successfully established.TLR5 mRNA expression level and CBLB502 activation from 2325 sequence infected cells were down-regulated to 0.32 times and 0.36 times normal control respectively.Conclusions Lentivirus particles interfering TLR5 which can be used to establish TLR5 stably down-regulated cell lines are successfully constructed.