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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。
Objective To prepare lentiviral interference particles of Toll-like receptor 5 (TLR5) and establish a HEK293 cell line that down-regulated MyD88. Methods DNA fragments carrying different specific interference sequences were inserted into the interference plasmid pSicoR and lentivirus particles were prepared. Lentiviral particles were transfected into HEK293 cells to establish a stable cell line. The down-regulation of TLR5 expression and the blockade of signal pathway initiation by different interference sequences were determined by RT-PCR and reporter gene analysis. Results Lentiviral particles with interference sequence were successfully prepared and HEK293 cell line stably infected with lentivirus was established. Compared with the control group, the level of TLR5 mRNA expression in cells infected with 2325 sequence was reduced to 0.32, and the activation of CBLB502 decreased to 0.36. Conclusion The TLR5 lentiviral interference particles were successfully prepared and a stable down-regulated TLR5 cell line was established.