巨噬细胞LRG47/EBP50真核共表达质粒的构建及表达鉴定

Construction and Identification of LRG47/EBP50 Coexpression Plasmid

目的:构建能共表达LRG47和EBP50的重组质粒pBud-LE,实现目的基因LRG47、EBP50在真核细胞中的表达,以便于后续从分子和细胞水平研究其抗结核分枝杆菌(Mtb)感染的效果和机制。方法:用RT-PCR方法从RAW264.7细胞中扩增获得小鼠的lrg47基因和ebp50基因,分步克隆入真核共表达质粒pBUDCE4.1中,构建真核共表达质粒pBud-LE。同时构建ebp50和红色荧光蛋白表达基因(rfp)的融合基因,置换pBud-LE中的ebp50基因,构建共表达LRG47和EBP50-RFP的重组质粒pBud-LER;将pBud-LE、pBud-LER通过脂质体转染入293T细胞,以pBud-LER转染观察转染效果,再分别采用RT-PCR和免疫印迹法鉴定质粒在真核细胞系中表达LRG47和EBP50的能力。结果:成功构建了真核共表达质粒pBud-LE和pBud-LER;转染293T细胞后,LRG47和EBP50在RNA水平和蛋白水平都明显升高。结论:成功的构建了真核共表达质粒pBud-LE。

Objective： To construct the eukaryotic co-expression plasmid encoding mouse EBP50 and LRG47 in eukaryotic cell for future exploration of therapeutic vaccines against tuberculosis.Methods： The coding sequence of EBP50 and LRG47 were amplified from the total RNA of RAW264.7 cells by RT-PCR and cloned into the eukaryotic coexpression plasmid pBudCE4.1 step-by step to form the recombinant plasmid pBudCE4.1-LRG47-EBP50（pBud-LE）.On the other hand,the fusion gene encoding EBP50 and red fluorescence protein（RFP） was constructed and inserted into pBud-LE to replace EBP50 coding sequence,to obtain the recombinant plasmid pBudCE4.1-LRG47-EBP50-RFP（pBud-LER）.pBud-LE and pBud-LER were transfected into 293T cells by LipofectamineTM2000.Transfection effect was evaluated by pBud-LER transfection by fluorescence microscope observation.The expression of LRG47 and EBP50 were detected by RT-PCR and Western-blot.Results： Restricted enzyme digestion and DNA sequencing showed that recombinant plasmids pBud-LE and pBud-LER were constructed successfully.The expression of EBP50 and LRG47 in pBud-LE transfected 293T cells were affirmed by both RT-PCR and Western blot.Conclusion： The eukaryotic coexpression plasmid encoding LRG47 and EBP50 was constructed successfully.