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目的制备MyD88分子的慢病毒干涉颗粒,建立稳定下调MyD88的HEK293细胞系。方法将携带特异性干涉序列的DNA片段克隆入干涉质粒pSicoR中,并制备携带不同干涉序列的慢病毒颗粒。将慢病毒颗粒感染HEK293细胞,建立稳定细胞系,经RT-PCR和报告基因分析方法确定不同干涉序列对MyD88表达的下调及信号通路激活的阻断作用。结果成功制备携带干涉序列的慢病毒颗粒,并获得稳定感染慢病毒的HEK293细胞系,经鉴定发现感染912位序列的细胞MyD88mRNA表达水平下调为对照组的28%,对CBLB502的激活作用下降为对照组的24%。结论成功制备MyD88的慢病毒干涉颗粒,可用于建立MyD88稳定下调的细胞系。
Objective To prepare lentiviral interference particles of MyD88 and establish a HEK293 cell line stably down-regulated MyD88. Methods DNA fragments carrying specific interference sequences were cloned into the interference plasmid pSicoR and lentivirus particles carrying different interference sequences were prepared. Lentiviral particles were transfected into HEK293 cells to establish stable cell lines. The down-regulation of MyD88 expression and the blockade of signal pathway activation 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 obtained. The expression of MyD88 mRNA in 912-infected cells was found to be down-regulated to 28% of the control group, and the activation of CBLB502 was reduced to the control 24% of the group. Conclusion The MyD88 lentiviral interference particles were successfully prepared and could be used to establish a stable MyD88 down-regulated cell line.