人PD-L1真核表达载体的构建及结肠癌细胞稳定表达单克隆株的筛选和鉴定

目的:构建人程序性死亡配体1（PD-L1）重组质粒载体，筛选稳定高表达人PD-L1的结肠癌单克隆细胞株。方法:提取HCT116细胞总RNA，RT-PCR克隆PD-L1基因片段，将其重组至含有HA标签和Neo真核细胞筛选抗性的pcDNA3质粒载体上，转染HCT116细胞，倍比稀释8个细胞浓度至96孔板，G418筛选两周，挑取单细胞克隆，通过免疫印迹鉴定外源性PD-L1的表达；通过MTS、Transwell和软琼脂细胞克隆形成实验比较PD-L1低表达和高表达单克隆细胞株生长增殖、迁移和细胞克隆形成能力的差别；最后通过在两组细胞中过表达帽依赖性荧光素酶报告基因阐述造成上述表型的可能机制。结果:成功构建人PD-L1的真核表达载体，并筛选获得稳定高表达PD-L1的HCT116单克隆细胞株。功能研究初步证明PD-L1可促进细胞克隆形成能力、迁移和生长增殖。过表达PD-L1可使帽依赖性荧光素酶报告基因的表达上调约3~4倍，表明其机制部分为PD-L1可上调帽依赖性蛋白质翻译。结论:获得了PD-L1重组质粒载体及可用于后续功能研究的稳定高表达人PD-L1的HCT116单克隆细胞株。

Construction of human PD-L1 eukaryotic expression plasmid and screening of single overexpression stable colon cancer cell line

Objective:To construct the human PD-L1 recombinant plasmid vector and screen the single stable colon cancer cell line over-expressing human PD-L1.Methods:Human PD-L1 gene was cloned by RT-PCR from total RNA in HCT116 cells.Gene fragments were linked to HA labeled pcDNA3 with Neo selecting marker to transfect HCT116 cells.Cells were then dispersed to 96-well plates and screened by G418 for 2 weeks.PD-L1 expression level in the monoclone cell lines were identified by Western Blot.The effects of PD-L1 expression level on cell growth migration and colony formation ability were evaluated by MTS，Transwell and soft agar assay.We further elucidated the partial mechanism leading to those phenomena by overexpression of cap-dependent luciferase reporter gene assay.Results:The human PD-L1 eukaryotic expression vector and monoclone PD-L1 over-expressing stable cell lines were successfully constructed.Preliminary function study showed that PD-L1 could promote colon cancer cell growth,migration and colony formation capacity.PD-L1 upregulated cap-dependent luciferase expression by 3~4 folds which indicated that the mechanism was partially from elevated cap-dependent protein expression in the PD-L1 high-expression cell lines.Conclusion:We generated the human PD-L1 recombinant plasmid vector and single HCT116 stable cell line over-expressing human PD-L1 which can be used for further PD-L1 functional study.