人PD-L1Ig融合蛋白的表达及其生物学活性的研究

Expression of Human PD-L1Ig Fusion Protein and Characteristics of Its Bioactivity

为获取人PD L1Ig融合蛋白 ,研究其对T细胞的调节效应 ,采用PCR法从pMD18 T/PD L1中扩增出人PD L1基因的胞外段序列 ,从人脾脏细胞中扩增出人IgG1Fc恒定区基因 ,两者拼接后插入逆转录病毒载体pEGZ Term ,用脂质体法与两个辅助病毒载体共转染 2 93T包装细胞 ,用含病毒颗粒的培养上清反复感染L92 9细胞 ,Zeocin筛选能稳定分泌人PD L1Ig蛋白的基因转染细胞并亚克隆之 ,经无血清培养 ,收集的上清用Dotblot检测、ELISA定量后 ,浓缩并经ProteinG柱纯化 ,再经Westernblot鉴定。以FACS分析纯化蛋白对活化T细胞表达的PD 1结合能力 ,以体外T细胞活化体系观察其对T细胞表型的调节作用。结果表明 ,成功地构建了表达人PD L1Ig蛋白的重组逆转录病毒载体 ;获得的L92 9/PD L1Ig细胞能稳定分泌人PD L1Ig蛋白 ;该蛋白与PD 1受体具有良好的结合能力 ,能抑制T细胞的进一步活化。

To stably express human PD-L1-Fc fusion protein in eukaryon cells and to investigate the regulatory effet on T cells. Extracellular domain of human PD-L1 gene was amplified by PCR from pMD18-T/PD-L1 vector, and human IgG1(Fc)constant region was obtained through RT-PCR from human spleen cells. Then, assembled by TP-PCR, the derived fragment consisting of the PD-L1ECD and Fc (PD-L1Ig) was inserted into retro virus vector pEGZ-Term to construct the recombinant vector pGEZ-Term/PD-L1Ig. The recombinant vector together with its 2 helper virus vectors was cotransfected into the package cell 293T. The supernatant of 293T containing intact virus granules was used to infect L929 cells. The FCS-free supernatant of infected L929 cells was harvested for subsequent analysis. Confirmed by Dot blot、ELESA and Western blot analysis, the condensed fusion protein was purified through protein G affinity chromatography column. T cells were incubated for 3 days in the wells of plates precoated with PD-L1Ig fusion protein and anti-CD3 mAb to detect its influence on the phenotypes of CD4+ and CD8+ T cells. It was found that recombinant retro virus vector carring human PD-L1Ig fusion gene was constructed successfully and L929/PD-L1Ig cells stably secreting human PD-L1Ig fusion protein were obtained and confirmed. FACS analysis showed its high affinity with its receptor, and the activation of T cells was inhibited obviously by PD-L1Ig in vitro. The construction of cells stably secreting human PD-L1Ig fusion protein and the prepartion of purified protein will contribute to further research on the functions of the gene.