人PD-L1真核表达载体的构建及其在HEK293细胞系中的稳定转染

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。

Construction of Eukaryotic Expressing Vector of Human PD-L1 and Establishment of Stable Transfectant HEK293 Cell Line

Objective To construct eukaryotic expressing vector of human PD-L1 gene and transfect HEK293 cells so as to establish stable HEK293 cell line. Methods PD-L1 gene was amplified by polymerase chain reaction from the human heart cDNA library and confirmed by DNA- sequence analysis. The PD-L1 gene was then digested with the restriction endonucleases Xhol and EcoR1 and inserted into eukaryotic expressing vector pIRES2-EGFP, pIRES2-EGFP/PD-L1 was transfected into HEK293 cells by lipofectamineTM2000. After screening culture by G418, stable transfected HEK293 cells line was established, and the expression of PD-L1 was identified by FCM, RT-PCR and Western blot. Results The eukaryotie expressing vector pIRES2-EGFP/PD-L1 was constructed, stable transfected HEK293 cell line was established, and PD-L1 gene was expressed successfully. Conclusion The construction of eukaryotic expressing vector pIRES2-EGFP/PD-L1 and the establishment of stable transfected HEK293 cell line have provided solid experiment foundation for further studies on the function of PD-L1.