Dnd1慢病毒表达载体的制备及鉴定

Construction and Identification of Lentiviral Expression Vector Carrying Dnd1

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 a lentiviral expression Vector Carrying Dnd1 and obtain Dnd1 efficient and stable expression in mammalian cells.Methods The DNA fragment of Dnd1 coding sequence was amplified by PCR with designed primer from the plasmid of pcDNA3.1-Dnd1,and then subcloned into pGC-FU vector with In-Fusion technique to generate the lentiviral expression vector,pGC-FU-Dnd1.The positive clones were screened by PCR and the correct Dnd1 was confirmed by sequencing.Recombinant lentiviruses were produced in 293T cells following the cotransfection of pGC-FU-Dnd1,and packaging plasmids of pHelper 1.0 and pHelper 2.0.Dnd1 protein expression in 293T cells was detected by Western blot.Results The lentiviral expression Vector Carrying Dnd1 was successfully constructed and Dnd1 protein expression was detected in 293T cells.Conclusion The recombinant lentiviruse pGC-FU-Dnd1 was successfully produced and it can establish a foundation for our study the molecular function of Dnd1.