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中文摘要：
目的：构建负载人恶性黑素瘤相关抗原gp100抗原肽的HLA-A^＊2402四聚体并进行鉴定,为分析gp100抗原肽诱发的特异性细胞免疫应答创造条件。方法：以羧基端融合生物素化酶BirA底物肽的HLA-A^＊2402重链胞外域融合蛋白、人β2-微球蛋白、gp100 VYFFLPDHL抗原肽、PE-链亲和素为材料,制备HLA-A^＊2402/VYF四聚体;非还原SDS-PAGE鉴定四聚体四聚化程度;三色免疫荧光标记流式细胞术分析HLA-A^＊2402＋健康志愿者外周血gp100特异性CD8＋T细胞的频率。结果：HLA-A^＊2402/VYF四聚体形成率为83%,所制备的四聚体具有与HLA-A^＊2402＋供者抗原特异性CD8＋T细胞的结合活性,特异性CD8＋T细胞的频率为外周血总CD8＋T细胞的0.02%～0.06%。结论：成功制备HLA-A^＊2402/VYF四聚体并用于检测gp100特异性CD8＋T细胞,为研究HLA-A^＊2402＋黑素瘤患者免疫治疗的机制及疗效监测创造了条件。
英文摘要：
Objective To prepare and verify the HLA-A^＊2402 tetramer loaded with melanoma associated antigen gp100 peptide.Methods HLA-A^＊2402-BSP,i.e.the ectodomain of HLA-A^＊2402 fused to a BirA substrate peptide（BSP） was refolded in the presence of human β2-microglobulin as well as the gp100 peptide（VYFFLPDHL,VYF）.The biotinylated HLA-A^＊2402/VYF molecules were purified,then bound to streptavidin-PE and tetramerized.The extent of tetramerization was detected by SDS-PADE under non-reducing conditions without boiling the sample.Finally,the prepared HLA-A^＊2402/VYF tetramer reagent was verified by detecting the CD8^＋ T cells specific for gp100 in HLA-A^＊2402 positive healthy volunteers by three-color flow cytometry.Results HLA-A^＊2402/VYF tetramer was successfully prepared.The extent of tetramerization was about 83%,and the tetramer positive rates were 0.02% to 0.06% within the CD8^＋ T cell populations in the peripheral blood of healthy HLA-A^＊2402 positive healthy volunteers.Conclusion The regent will facilitate the study of the immune responses of gp100 VYF-specific CD8^＋ T cells in the clinical trials of immunotherapy on melanoma patients with HLA-A^＊2402 allele.
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