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目的:构建并制备携带靶向干扰Notch-1基因shRNA的重组腺相关病毒载体。方法:设计针对Notch-1的靶DNA序列,构建重组质粒pAAV-MCS2/siRNA-Notch-1,将该质粒和腺相关病毒包装质粒pAAV-RC,腺病毒辅助质粒pHelper共转染QBI-293A细胞,包装成带有Notch-1基因的重组腺相关病毒。结果:PCR鉴定分析结果表明成功构建针对Notch-1的siRNA重组腺相关病毒载体,滴定法测定重组病毒载体基因组得到滴度为1.2×1012VP/L的高滴度腺相关病毒。结论:成功构建携带Notch-1基因短发夹状干扰RNA的腺相关病毒载体,为探索Notch-1基因在胶质母细胞瘤肿瘤干细胞中的作用提供实验基础。
OBJECTIVE: To construct and prepare a recombinant adeno-associated virus vector carrying shRNA targeting Notch-1 gene. METHODS: The target DNA sequence of Notch-1 was designed and the recombinant plasmid pAAV-MCS2 / siRNA-Notch-1 was constructed. The plasmid and adeno-associated virus packaging plasmid pAAV-RC and adenovirus helper plasmid pHelper were co-transfected into QBI-293A cells , Packaged as a recombinant adeno-associated virus carrying the Notch-1 gene. Results: The results of PCR analysis showed that siRNA recombinant adeno-associated virus vector targeting Notch-1 was successfully constructed and the titer of recombinant adenoviral vector was 1.2 × 1012VP / L. Conclusion: The adeno-associated virus vector carrying short hairpin RNA of Notch-1 gene was successfully constructed, which provided the experimental basis for exploring the role of Notch-1 gene in glioblastoma tumor stem cells.