核糖体蛋白S3a RNA干扰重组慢病毒载体的构建及其对细胞凋亡的影响

目的 构建表达小鼠核糖体蛋白S3a 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RPS3a mRNA 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 pLLU2G-eGFP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Western blot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Lenti-shmRPS3a慢病毒载体,病毒滴度为6-9×10₇TU/ml；RT-PCR表明Lenti-shmRPS3a的沉默效率高达72.64%、Western Blot证明RPS3a蛋白水平表达降低；流式细胞术证明感染了Lenti-shmRPS3a慢病毒载体的细胞凋亡率较对照组明显升高。结论 表达小鼠核糖体蛋白S3a shRNA慢病毒载体能有效沉默RPS3a基因表达,促进细胞凋亡。

Construction of recombinant lentiviral vector harboring shRNA of ribosomal protein S3a and its effect on cell apoptosis

Objective To construct a recombinant lentiviral vector harboring shRNA for mouse ribosomal protein S3a (RPS3a) and to analyze its effect on cell apoptosis. Methods Four pairs of shRNA sequences targeting mouse RPS3a mRNA were designed, and were ligated into pLLU2G-eGFP lentiviral vector. The recombinant plasmids were co-transfected with pLV/helper plasmids into 293T cells to package the recombinant lentivirus and the titers of the virus were determined. The lentivirus was introduced into RAW264.7 cells and levels of RPS3a mRNA and protein were detected by real-time PCR and Western blotting analysis, respectively. The apoptosis of RAW264.7 cells was detected by flow cytometry assays. Results PCR and DNA sequencing analysis confirmed that the recombinant lentivirus was successfully constructed and the virus titer was 6×10⁷-9×10⁷ TU/mL. Results of real-time PCR showed that the silencing efficiency of Lenti-shmRPS3a was 72.64%, and Western blotting analysis showed that RPS3a protein expression was decreased. Flow cytometry demonstrated that lentiviral-shRPS3a significantly increased cell apoptosis compared with the control group (P<0.05). Conclusion The constructed lentiviral vector harboring shRNA of RPS3a can efficiently silence RPS3a gene expression and promote cell apoptosis.