| 赛佳明 马学晓 邱晨生 陈伯华△ 胡有谷.慢病毒GV115-AIF siRNA 重组表达系统的构建及鉴定[J].,2016,16(9):1636-1638 |
| 慢病毒GV115-AIF siRNA 重组表达系统的构建及鉴定 |
| Construction and Detection of GV115-AIF siRNA |
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| DOI： |
| 中文关键词: 慢病毒 凋亡诱导因子(AIF) RNA干扰 基因治疗 |
| 英文关键词: Lentivirus Apoptosis inducing factor (AIF) RNA interference (RNAi ) Gene therapy |
| 基金项目:国家自然科学基金项目（81171758） |
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| 中文摘要: |
| 目的：构建高效的慢病毒GV115-AIF siRNA重组表达系统。方法：根据目的基因AIF以及RNA 干扰序列设计原则，利用设
计软件设计了3 个可能的AIF siRNA 序列。应用全基因合成技术和亚克隆技术构建GV115-AIF siRNA重组质粒，并采用聚合酶
链反应技术（PCR 技术）和基因测序技术对GV115-AIF siRNA 重组质粒鉴定。利用GV115 病毒包装辅助pHelper 1.0 质粒和
pHelper 2.0 质粒进行病毒包装。病毒包装后转染人胚肾293T细胞，通过应用逆转录定量PCR技术（RT-PCR 技术）检测转染后对
人胚肾293T 细胞中AIF基因的敲减效率，筛选最高效的AIF siRNA 基因序列。结果：GV115-AIF siRNA 质粒PCR 鉴定显示位于
341bp 附近的条带。测序结果与设计的基因序列完全吻合。3 个可能的AIF siRNA 序列中基因敲减效率最高的可达到88.3%。结
论：成功构建高效的慢病毒GV115-AIF siRNA重组表达系统。 |
| 英文摘要: |
| Objective:To construct and detect the GV115-AIF siRNA.Methods:On the basis of AIF gene sequence or RNAi design
principle, three AIF siRNA sequence were designed by design software. The recombinant plasmid 0f GV115-AIF siRNA was constructed
by gene synthesis and subclone technique. The GV115-AIF siRNA was detected by Polymerase Chain Reaction (PCR) and DNA
sequencing. The GV115 virus was packaged adopting pHelper 1.0 plasmid or pHelper 2.0 plasmid. After the lentivirus had been packaged,
the 293T cells were transfected by GV115-AIF siRNA. The translation of AIF gene transfected by GV115-AIF siRNA was detected
using reverse transcription-polymerase chain reaction (RT-PCR), and the most effective gene sequence of AIF siRNA was screened.Results:The GV115-AIF siRNA was proved to be right using PCR and DNA sequencing. The gene knocking rate of the most efficient
GV115-AIF siRNA was 88.3%.Conclusion:The GV115-AIF siRNA was constructed successfully. |
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