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目的构建小鼠核糖体蛋白S3a(RPS3a)特异性RNA干扰重组慢病毒载体(Lenti-shmRPS3a),分析其对细胞凋亡的影响。方法设计针对小鼠RPS3amRNA的4条干扰序列,合成相应的发夹序列,连接入慢病毒载体系统pLLU2GeGFP中构建重组质粒,将重组质粒与辅助包装质粒共转染293T细胞组装病毒,检测病毒滴度。病毒感染RAW264.7细胞后,RT-PCR和蛋白质印迹法检测干扰效果,流式细胞术分析其对凋亡的影响。结果 PCR和测序证明成功构建出LentishmRPS3a慢病毒载体,病毒滴度为(6~9)×107 TU/mL;RT-PCR表明Lenti-shmRPS3a的沉默效率高达72.64%,蛋白质印迹法证明RPS3a蛋白表达水平降低;流式细胞术证明感染了Lenti-shmRPS3a的细胞凋亡率较对照组升高(P<0.05)。结论表达小鼠RPS3ashRNA的慢病毒载体能有效沉默RPS3a基因表达,促进细胞凋亡。
Objective To construct the Lenti-shmRPS3a RNAi-specific RNA interference expression vector of murine ribosomal protein S3a (RPS3a) and analyze its effect on apoptosis. Methods Four interfering sequences of mouse RPS3amRNA were designed and the corresponding hairpin sequences were synthesized and ligated into the lentiviral vector system pLLU2GeGFP to construct a recombinant plasmid. The 293T cells were co-transfected with the recombinant plasmids and the auxiliary packaging plasmid to detect the virus titer . After infection with RAW264.7 cells, the interference effect was detected by RT-PCR and Western blotting, and the effect of apoptosis was analyzed by flow cytometry. Results The LentishmRPS3a lentiviral vector was successfully constructed by PCR and sequencing. The virus titer was (6 ~ 9) × 107 TU / mL. RT-PCR showed that the silencing efficiency of Lenti-shmRPS3a was as high as 72.64%. Western blot showed that RPS3a protein expression The apoptosis rate of Lenti-shmRPS3a infected cells was higher than that of the control group (P <0.05). Conclusion The lentiviral vector expressing mouse RPS3ashRNA can effectively silence RPS3a gene expression and promote apoptosis.