中国肿瘤生物治疗杂志2019，Vol.26Issue(4)：389-395,7.DOI:10.3872/j.issn.1007-385x.2019.04.004
PD-S15融合蛋白体外特异性靶向PD-1分子并快速扩增NK/T细胞
PD-S15 fusion protein specifically target PD-1 and rapidly expands NK/T cells
摘要
Abstract
Objective: To investigate the function of anti-PD-1 (scFv) /IL-15/IL-15 Rα-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-15 Rα-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 HEK293 T cells by lipofectamineTM2000. 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 HEK293 T 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.关键词
程序性死亡受体1/白细胞介素-15/白细胞介素-15受体/PD-S15融合蛋白/过继细胞治疗/抗原特异性T细胞Key words
programmed death receptor-1 (PD-1)/IL-15/IL-15Rα/PD-S15 fusion protein/adoptive cell therapy/antigen-specific T cell分类
医药卫生引用本文复制引用
王阿香,李铁鹏,张放,王瑶,杜雪相,高全立..PD-S15融合蛋白体外特异性靶向PD-1分子并快速扩增NK/T细胞[J].中国肿瘤生物治疗杂志,2019,26(4):389-395,7.基金项目
国家自然科学基金资助项目(No.81502468) （No.81502468）
河南省医学科技攻关计划项目(No.201701030) （No.201701030）
河南省自然科学基金项目(No.182300410344) （No.182300410344）
河南省科技攻关计划项目(No.162300410095) （No.162300410095）
