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目的:构建小鼠Dnd1慢病毒表达载体,以期在哺乳动物细胞中高效、稳定表达。方法:设计引物引入AgeⅠ酶切位点,使用PCR方法从质粒pcDNA3.1-Dnd1中扩增小鼠Dnd1基因的编码区序列,对所扩增出的目的片段回收纯化。用In-Fusion技术将AgeⅠ内切酶消化后目的片段交换连接入AgeI酶切的pGC-FU载体,构建Dnd1慢病毒表达载体pGC-FU-Dnd1。酶切验证并测序正确后,将质粒pGC-FU-Dnd1与慢病毒辅助包装载体共转染293T细胞,Western-blot验证Dnd1在转染293T细胞中表达。结果:通过PCR扩增获得了Dnd1基因,将Dnd1克隆到慢病毒转移质粒pGC-FU中,并在293T细胞中包装产生慢病毒颗粒。结论:成功构建了Dnd1慢病毒表达载体,为进一步从分子水平探讨Dnd1功能奠定了基础。
Objective: To construct the mouse Dnd1 lentivirus expression vector with high efficiency and stable expression in mammalian cells. Methods: The primers designed to introduce Age Ⅰ restriction sites were used to amplify the coding region of mouse Dnd1 gene from plasmid pcDNA3.1-Dnd1 by PCR. The target fragment was recovered and purified. The gene fragment digested with AgeⅠ endonuclease was ligated into the pGC-FU vector digested by AgeI using In-Fusion technology to construct the Dnd1 lentivirus expression vector pGC-FU-Dnd1. After restriction endonuclease digestion and sequencing, 293T cells were co-transfected with plasmid pGC-FU-Dnd1 and lentivirus helper vector, and Dnd1 expression in transfected 293T cells was confirmed by Western-blot. Results: The Dnd1 gene was obtained by PCR amplification, Dnd1 was cloned into lentiviral transfer plasmid pGC-FU and packaged in 293T cells to produce lentivirus particles. Conclusion: The Dnd1 lentiviral vector was constructed successfully, which laid the foundation for the further exploration of Dnd1 function at the molecular level.