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  

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

作　　者：朱云霞[1] 胡振华[2] 陈永井[2] 李文香[2] 陈成[1] 黄建安[1] 张学光[2] 

机构地区：[1]苏州大学附属第一医院呼吸内科,江苏苏州215006 [2]苏州大学医学生物技术研究所,江苏苏州215007

出　　处：《苏州大学学报（医学版）》2009年第3期459-462,496,共5页Suzhou University Journal of Medical Science

基　　金：国家自然科学基金资助项目(30770947);江苏省临床免疫学重点实验室基金资助项目

摘　　要：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 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.

关 键 词：PD-L1基因 真核表达载体 转染 HEK293细胞 

分 类 号：R392.2[医药卫生—免疫学]