Li Da-Wei, Yu Jian-Feng, Chen Yong-Jing, Ma Hong-Bing, Wang Zheng-Fei, Zhu Yi-Bei, Zhang Xue-Gang
Biotechnology Research Institute, Soochow University, Suzhou 215007, China.
Acta Biochim Biophys Sin (Shanghai). 2004 Feb;36(2):141-6. doi: 10.1093/abbs/36.2.141.
Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed on activated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibits proliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involved in antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain was amplified and cloned into expression plasmid pGEX-5x-3. The fusion protein GST-PD-1 was effectively expressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was performed to obtain bioactive soluble GST-PD-1. Fusion protein of above 95% purity was acquired by a convenient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependent in vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-PD-1 could competently block the interaction between PD-L1 and PD-1 and increase the production of IL-2 and IFN-gamma of phytohemagglutinin-activated T cells.
程序性死亡蛋白 1（PD-1）是一种表达于活化的 T 细胞、B 细胞和髓样细胞上的 CD28 家族共刺激分子。PD-1 与其两种配体 PD-L1 和 PD-L2 的结合会抑制 T 细胞增殖及其一系列细胞因子的产生。PD-1 通路的阻断参与抗病毒和抗肿瘤免疫。在本研究中，编码细胞外结构域的人 PD-1 cDNA 被扩增并克隆到表达质粒 pGEX-5x-3 中。融合蛋白 GST-PD-1 在大肠杆菌 BL21(DE3)中以包涵体形式有效表达，并进行了变性和复性操作以获得具有生物活性的可溶性 GST-PD-1。通过使用 GST 亲和柱和尺寸排阻柱进行便捷的两步纯化，获得了纯度高于 95%的融合蛋白。此外，建立了一种依赖 PD-L1 的体外生物测定方法来表征 GST-PD-1 的生物活性。结果表明，GST-PD-1 能够有效阻断 PD-L1 与 PD-1 之间的相互作用，并增加植物血凝素激活的 T 细胞中白细胞介素-2（IL-2）和干扰素-γ（IFN-γ）的产生。
