[摘要] 目的：探讨anti-PD-1（scFv）/IL-15/IL-15Rα-sushi （简称PD-S15）融合蛋白体外特异性结合PD-1 的能力及其对NK/T细胞增殖能力的影响。方法：化学合成人anti-PD-1（scFv）基因和人IL-15/IL-15Rα-sushi 融合基因，经过酶切连接构建重组表达质粒pUC57-PD-S15，用LipofectamineTM 2000 瞬时转染HEK293T细胞，收获细胞培养液上清，用Wb法检测细胞培养液上清中PDS15融合蛋白的表达；用不同配比的PD-S15/X-VIVOTM15 培养液对PBMC和TIL 分别诱导培养后，用流式细胞术检测PD-S15 融合蛋白体外特异性结合PD-1 的能力和对PBMC增殖及CD3+CD8+、CD3+CD4+、CD3-CD56+各细胞亚群比例的影响；用细胞计数法检测PD-S15 融合蛋白对TIL 增殖能力的影响。结果：pUC57-PD-S15 表达质粒经双酶切和测序验证构建成功并成功转染HEK293T细胞，目标蛋白相对分子质量大约为55 000，符合预期。PD-S15 融合蛋白体外具有PD-1 特异性结合能力（P<0.05）及NK/T细胞活化增殖能力（P<0.05）；与经典TIL 培养方案相比，PD-S15 培养方案体外活化扩增T细胞的能力更强（P＜0.01）。结论：PD-S15 融合蛋白体外能够特异性靶向PD-1 分子并快速扩增NK/T细胞，为后续从肿瘤组织内或外周血中选择性扩增CD8+PD-1+抗原特异性T细胞奠定了基础。

[Abstract] Objective: To investigate the function of anti-PD-1 (scFv)/IL-15/ IL-15Rα - sushi fusion protein (PD-S15) to specifically bind to PD-1 in vitro and to explore its effect on NK/T cell proliferation. Methods: The human anti-PD-1 (scFv) gene sequence and human IL-15/IL-15Rα-sushi fusion gene sequence were synthesized chemically. The recombinant expression plasmid pUC57-PD-S15 was constructed by enzyme digestion and ligation of the two target genes, and then transiently transfected into HEK293T cells by lipofectamineTM 2000. The supernatants of cell culture medium were acquired, and the expression of PD-S15 fusion protein in cell culture supernatants was detected by Wb assay. PBMCs and TILs were cultured in mediums with different proportion of PD-S15/X-VIVOTM15,respectively. Then, the capacity of PD-S15 fusion protein to bind to PD-1 in vitro and its effect on the proliferation of PBMCs and the proportion of CD3+CD8+, CD3+CD4+ and CD3-CD56+ subsets were detected by flow cytometry. The effect of PD-S15 fusion protein on the proliferation of TILs was detected by cytometry. Results: The successful construction of pUC57-PD-S15 eukaryotic expression plasmid was confirmed by double enzyme digestion and sequencing, and then successfully transfected into HEK293T cells. The relative molecular weight of the target protein was approximately 55 000, and was in line with expectations. PD-S15 fusion protein could specifically combine with PD-1 in vitro (P<0.05) and stimulate NK/T cell proliferation (P<0.05). Compared with classical TILs culture method,the efficiency of activation and amplification of T cells in vitro by PD-S15 culture method was better (P<0.01). Conclusion: PD-S15 fusion protein can specifically target PD-1 and rapidly expand NK/T cells in vitro, which lays a foundation for the selective expansion of CD8+PD-1+ antigen-specific T lymphocytes from tumor tissues and even peripheral blood.

国家自然科学基金资助项目（No.81502468）；河南省医学科技攻关计划项目（No.201701030）；河南省自然科学基金项目（No. 182300410344）；河南省科技攻关计划项目（No. 162300410095）