| 摘要: |
| 【目的】构建包含Sf-caspase-1反向重复序列的重组杆状病毒vAcMNPV-dsCasp，并在草地贪夜蛾(Sf9)细胞中表达Sf-caspase-1双链RNA，用以抑制Sf9细胞的凋亡，为未来优化杆状病毒表达系统提供试验依据。【方法】将Sf-caspase-1反向重复序列构建到pBac5上，与AcMNPV Bacmid共转染Sf9细胞产生重组杆状病毒，用RT-PCR和PI活细胞染色方法，分别检测Sf-caspase-1 mRNA含量及Sf9细胞的凋亡情况。【结果】PCR和双酶切检测结果表明，成功构建了重组质粒pBac5-dsCasp，并得到重组杆状病毒vAcMNPV-dsCasp。PI染色结果表明，相比野生型病毒vAcMNPV,重组杆状病毒vAcMNPV-dsCasp能够抑制Sf9细胞的凋亡。RT-PCR结果表明，vAcMNPV-dsCasp感染的Sf9细胞中Sf-caspase-1 mRNA含量明显下降。【结论】在重组杆状病毒上表达Sf-caspase-1双链RNA确实能够沉默Sf-caspase-1，从而抑制细胞凋亡。 |
| 关键词: vAcMNPV RNAi dsRNA Sf-caspase-1 细胞凋亡 杆状病毒表达系统 |
| DOI： |
| 分类号: |
| 基金项目:陕西省自然科学基金项目（2011JM3002） |
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| Influence of insect cell apoptosis by expression of Sf-caspase-1 double stranded RNA in recombinant baculovirus |
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OU Yanmei,XU Xiaodong
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| Abstract: |
| 【Objective】This study constructed recombinant baculovirus containing inverted repeat sequence of Sf-caspase-1.The recombinant baculovirus expressed Sf-caspase-1 double-stranded RNA and inhibited apoptosis of Spodoptera frugiperda (Sf9) cell.【Method】Inverted repeat sequence of Sf-caspase-1 was inserted into pBac5 before being co-transfected with AcMNPV Bacmid into Sf9 cells to produce recombinant baculovirus.RT-PCR and PI vital cell staining were used to detect the content of Sf-caspase-1 mRNA and apoptosis of Sf9,respectively.【Result】PCR and restriction enzyme digestion showed that pBac5-dsCasp vector was built successfully.The recombinant virus vAcMNPV dsCasp was obtained by recombination between Bacmid and pBac5 dsCasp.Comparing with the cell infected by vAcMNPV,RT-PCR and PI vital cell staining indicated that vAcMNPV dsCasp inhibited cell apoptosis and the content of Sf-caspase-1 mRNA declined.【Conclusion】Expression of Sf-caspase-1 double-strained RNA in recombinant baculvirus silenced Sf-caspase-1,and inhibited cell apoptosis. |
| Key words: vAcMNPV RNAi dsRNA Sf-caspase-1 cell apoptosis baculovirus expression system |
