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

作　　者：赛佳明[1] 马学晓[1] 邱晨生 陈伯华[1] 胡有谷[1] 

机构地区：[1]青岛大学附属医院脊柱外科,山东青岛266003

出　　处：《现代生物医学进展》2016年第9期1636-1638,1653,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(81171758)

摘　　要：目的:构建高效的慢病毒GV115-AIF si RNA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 si RNA序列。应用全基因合成技术和亚克隆技术构建GV115-AIF si RNA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 si RNA重组质粒鉴定。利用GV115病毒包装辅助p Helper 1.0质粒和p Helper 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 si RNA基因序列。结果:GV115-AIF si RNA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 si RNA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 si RNA重组表达系统。Objective： To construct and detect the GV115-AIF si RNA. Methods： On the basis of AIF gene sequence or RNAi design principle, three AIF si RNA sequence were designed by design software. The recombinant plasmid 0f GV115-AIF si RNA was constructed by gene synthesis and subclone technique. The GV115-AIF si RNA was detected by Polymerase Chain Reaction（PCR） and DNA sequencing. The GV115 virus was packaged adopting p Helper 1.0 plasmid or p Helper 2.0 plasmid. After the lentivirus had been packaged, the 293 T cells were transfected by GV115-AIF si RNA. The translation of AIF gene transfected by GV115-AIF si RNA was detected using reverse transcription-polymerase chain reaction（RT-PCR）, and the most effective gene sequence of AIF si RNA was screened. Results： The GV115-AIF si RNA was proved to be right using PCR and DNA sequencing. The gene knocking rate of the most efficient GV115-AIF si RNA was 88.3%. Conclusion： The GV115-AIF si RNA was constructed successfully.

关 键 词：慢病毒 凋亡诱导因子(AIF) RNA干扰 基因治疗 

分 类 号：Q75[生物学—分子生物学] Q78