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目的构建含有人PD-L1基因的真核表达载体,并通过稳定转染获得稳定表达PD-L1分子的HEK293细胞系。方法从人心脏cDNA文库中扩增出PD-L1基因,通过双酶切(Xho1和EcoR1)装入真核表达载体pIRES2-EGFP中,脂质体法转染HEK293细胞,通过G418筛选,建立稳定高表达PD-L1分子的HEK293细胞系,通过流式细胞术、RT-PCR及Westernblot分析PD-L1的表达。结果构建了真核表达载体pIRES2-EGFP/PD-L1,建立了稳定转染PD-L1目的基因的HEK293细胞系。结论成功构建了PD-L1真核表达载体并获得了稳定转染该分子的HEK293细胞系。
Objective To construct eukaryotic expression vector containing human PD-L1 gene and obtain HEK293 cell line stably expressing PD-L1 by stable transfection. Methods PD-L1 gene was amplified from human cardiac cDNA library and inserted into eukaryotic expression vector pIRES2-EGFP by double enzyme digestion (Xho1 and EcoR1). The recombinant plasmid was transfected into HEK293 cells by Lipofectamine 2000, and then was screened by G418 to establish a stable HEK293 cell line highly expressing PD-L1 molecule, PD-L1 expression was analyzed by flow cytometry, RT-PCR and Western blotting. Results The eukaryotic expression vector pIRES2-EGFP / PD-L1 was constructed and HEK293 cell line stably transfected with PD-L1 gene was established. Conclusion The PD-L1 eukaryotic expression vector was successfully constructed and the HEK293 cell line stably transfected with this molecule was obtained.