[摘 要] 目的：基于噬菌体展示靶向锚定蛋白文库，以人白细胞抗原G（HLA-G）为靶点筛选锚定序列作为HLA-G结合蛋白 （HGBP）并评价其功能。方法：利用TCGA、GTEx等数据库分析肿瘤组织中HLA-G表达与临床预后和免疫浸润的相关性。利 用噬菌体展示靶向锚定蛋白文库对HLA-G胞外片段进行生物淘选并随机挑取单克隆进行测序。通过ELISA、免疫荧光染色鉴 定优势噬菌体克隆的功能。利用原核体系生产纯化HGBP，通过ELISA、表面等离子共振（SPR）、免疫荧光染色法评价其亲和力 及肿瘤特异性结合能力。结果：生物信息学分析发现，HLA-G在肿瘤组织中普遍呈高表达，其与临床总生存期、免疫细胞浸润水 平具有相关性（P < 0.05）。5轮噬菌体文库筛选后获得优势克隆，ELISA及免疫荧光染色结果均显示，优势噬菌体对HLA-G阳性 细胞结合显著强于阴性细胞（P < 0.05, P < 0.001）。经纯化生产的HGBP与HLA-G之间的亲和力可达17 nmol/L，ELISA结果显 示，HGBP与HLA-G分子的结合显著（P < 0.05）；免疫荧光染色结果表明，HGBP能与HLA-G阳性细胞特异性结合（P < 0.01）。 结论：经噬菌体展示文库筛选出的HGBP对HLA-G具有高亲和力，可特异性结合肿瘤细胞表达的HLA-G。

[Abstract] Objective: To identify HLA-G-binding proteins (HGBPs) by screening targeting ankyrin sequences from a phage display based ankyrin protein library using human leukocyte antigen G (HLA-G) as the target, and to evaluate their functions. Methods: The expression of HLA-G in tumor tissues and its correlation with clinical prognosis and immune infiltration were analyzed using bioinformatics tools such as TCGA and GTEx databses. The extracellular domain of HLA-G was subjected to biopanning with a phage displayed ankyrin protein library, followed by random selection and sequencing of monoclonal phage clones. The functional properties of dominant phage clones were validated using ELISA and immunofluorescence staining. HGBPs were produced and purified using a prokaryotic expression system, and their affinity and tumor-specific binding ability were evaluated using ELISA, surface plasmon resonance (SPR), and immunofluorescence staining. Results: Bioinformatics analysis revealed that HLA-G is widely overexpressed in tumor tissues and is correlated with overall survival (OS) and immune cell infiltration (P < 0.05). After five rounds of biopanning, dominant clones were obtained. Both ELISA and immunofluorescence staining results showed that these dominant phages had a significantly higher affinity to HLA-G positive cells compared to HLA-G negative cells (P < 0.05, P < 0.001). The purified HGBPs exhibited an affinity of up to 17 nmol/L for HLA-G. ELISA results showed significant binding of HGBP to HLA-G (P < 0.05), and immunofluorescence staining confirmed that HGBP could specifically bind to HLA-G-positive cells (P < 0.01). Conclusion: The HGBPs identified via phage display exhibit high affinity and specificity to HLA-G on tumor cells.

[基金项目] 国家自然科学基金（No. 82272811）