可溶性人sPD-L1及其抗体的制备和鉴定  

Preparation and Identification of Human Soluble sPD-L1 and Its Antibodies

作　　者：徐丽慧[1] 迟晓云[1] 李丰耀[1] 贾仟涛[1] 查庆兵[1] 何贤辉[1] 

机构地区：[1]暨南大学生命科学技术学院组织移植与免疫实验中心

出　　处：《生物工程学报》2007年第1期106-111,共6页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30572199和30371651)资助项目~~

摘　　要：通过固定化金属离子亲和层析进行柱上复性与纯化,获得高纯度的可溶性PD-L1胞外域(sPD-L1),其纯度达95%,纯化的sPD-L1经免疫印迹分析得到验证,并具有与其受体PD-1的特异性结合活性;以该抗原免疫小鼠获得高滴度的抗血清,并以制备的sPD-L1-HiTrap亲和层析柱纯化获得高纯度特异性抗体;将该抗体与另一商业化抗体结合建立了一种灵敏的双夹心ELISA法,检测范围为1ng/mL^100ng/mL,可用于分析可溶性PD-L1的含量。可溶性sPD-L1及其抗体的制备不仅可用于人体内特异性抗体和可溶性PD-L1的检测,同时也为进一步研究其体内外活性及其受体的性质提供了条件。This study reports the preparation and identification of soluble programmed death-1 （PD-1） ligand-1 （sPD-L1） and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95 % was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-L1 and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.

关 键 词：PD-L1 原核表达 重折叠 亲和层析 抗体 

分 类 号：Q789[生物学—分子生物学]