重组杆状病毒表达Sf-caspase-1双链RNA对昆虫细胞凋亡的影响

Influence of insect cell apoptosis by expression of Sf-caspase-1 double-stranded RNA in recombinant baculovirus

【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-1mRNA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-1mRNA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。

【Objective】This study constructed recombinant baculovirus containing inverted repeat sequence of Sf-caspase-1.The recombinant baculovirus expressed Sf-caspase-1double-stranded RNA and inhibited apoptosis of Spodoptera frugiperda（Sf9）cell.【Method】Inverted repeat sequence of Sf-caspase-1was 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-1mRNA 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 Sfcaspase1 mRNA declined.【Conclusion】Expression of Sf-caspase-1double-strained RNA in recombinant baculvirus silenced Sf-caspase-1,and inhibited cell apoptosis.