PD-L1抑制小鼠脾淋巴细胞增殖药物筛选模型的建立及应用

目的通过构建重组的小鼠pcDNA3.1(+)-PD-L1质粒,并在真核细胞中进行高表达,建立PD-L1抑制小鼠脾淋巴细胞增殖药物筛选模型用于免疫调节药物的筛选。方法以RT-PCR方法扩增得到小鼠的PD-L1全长片段;以pcDNA3.1(+)真核表达质粒为载体,构建得到重组质粒pcDNA3.1(+)-PD-L1;脂质体转染到小鼠成纤维细胞(L929),通过G418筛选得到高表达PD-L1的稳定细胞株并经免疫荧光鉴定;用丝裂霉素处理过的PD-L1高表达细胞与小鼠脾淋巴细胞共培养;成功建立PD-L1抑制小鼠脾淋巴细胞增殖的模型之后向其中加入不同种类、不同浓度的天然产物提取物,并设复孔。结果通过Eco RI/Xho I双酶切及测序证实构建的重组质粒pcDNA3.1(+)-PD-L1正确。RT-PCR和免疫荧光方法鉴定重组质粒在L929细胞中高效表达,应用流式细胞术成功建立PD-L1抑制小鼠脾淋巴细胞增殖的模型,并筛选出阻断PD-L1活性的有效药物。结论获得了稳定高表达PD-L1的细胞,并且用于免疫调节的药物筛选,为免疫调节药物的筛选奠定了实验基础。

The establishment of inhibitition of lymphocyte proliferation mediated by PD-L1 for drug screening

To express murine PD-L1 full length gene in L929 cells and investigate the role in lymphocyte proliferation,mPD-L1 full length gene was cloned and expressed.mPD-L1 full length gene was amplified from B16 tumor cell cDNA by RT-PCR and cloned into eucaryotic expression vector pcDNA3.1(+).Plasmid pcDNA3.1(+)-PD-L1 was transfected into L929 cells by lipofectamine,and the transfected cells were selected by G418.The PD-L1 expression in the transfected cells were detected by RT-PCR and immunocytochemistry.The bioa...