PD-1胞外段cDNA在真核细胞的表达与其功能鉴定  

Eukaryotic Expression and Functional Characterization of PD-1 Extracellular Domain

作　　者：贺宇飞[1] 张桂梅[1] 王小红[1] 张慧[1] 袁野[1] 李东[1] 冯作化[1] 

机构地区：[1]华中科技大学同济医学院生化与分子生物学系,武汉430030

出　　处：《生物工程学报》2004年第5期699-703,共5页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展 ( 973 )计划资助项目 (No .2 0 0 2CB5 13 10 0 )~~

摘　　要：PD L PD 1是参与肿瘤免疫逃避的一条抑制性信号途径。为了用可溶性的PD 1受体阻断PD L PD 1的相互作用 ,将小鼠PD 1胞外段 (aa1 aa16 7)作为独立可溶性分子进行了真核表达 ,并对其功能进行鉴定。构建了编码小鼠PD 1胞外段cDNA(sPD 1)的真核质粒表达载体pPD 1A和编码sPD 1 GFP重组蛋白的真核质粒表达载体pPD 1B ;细胞转染实验表明其表达产物主要是分泌到细胞外的可溶性产物 (sPD 1) ,流式细胞仪检测表明sPD 1可有效结合PD 1配体 ;肿瘤细胞杀伤实验表明 ,sPD 1作用于肿瘤细胞或在脾淋巴细胞激活过程中作用于淋巴细胞 ,均可增强Hsp70 H2 2抗原肽复合物激活的脾淋巴细胞杀伤肿瘤细胞的作用。sPD 1真核表达载体的构建为在肿瘤局部表达抑制性共刺激分子的可溶性受体 ,拮抗肿瘤微环境中负调节因素对T细胞的抑制作用 ,增强机体的抗肿瘤能力 。The negative signal provided by interactions of costimulatory molecules, programmed death 1 (PD 1) and its ligands, PD L1 (also B7 H1)and PD L2 (also B7 DC), is involved in the mechanisms of tumor immune evasion. To block PD Ls PD 1 interactions by a soluble receptor of PD 1, we constructed a eukaryotic expression plasmid that expresses extracellular region (aa1 aa167) of murine PD 1 (pPD 1A) and, another version of pPD 1A, pPD 1B, carrying cDNAs encoding for both extracellular region of PD 1 and green fluorescent protein (GFP) reporter gene, which was inserted downstream of PD 1 Experiment of BHK cells transfected with pPD 1B determined that most expression product (sPD 1) in the cells was secreted out. FACS analysis revealed that sPD 1 was specific and bound efficiently to PD 1 ligands. Cytotoxicity assay showed that blocking PD Ls on either tumor cells or spleen cells by sPD 1 mediated enhanced lysis of H22 cells by Hsp70 H22 peptides complex stimulated spleen cells. The constructed plasmid vector would provide a novel method of tumor gene therapy of blocking PD Ls PD 1 interactions by expression of soluble receptor of PD 1 in tumor sites, which could increase the antitumor activity.

关 键 词：T细胞 肿瘤 可溶性受体 真核表达 质粒载体 真核细胞 

分 类 号：R73-3[医药卫生—肿瘤]