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目的探讨纯化的隐球菌抗原肽GMFDGLSGV二聚体、四聚体、八聚体重组蛋白激发细胞毒性T淋巴细胞(Cytotoxic T lymphocyte,CTL)免疫应答的能力,为新型隐球菌疫苗的研制打下基础。方法通过细胞增殖和细胞毒性实验,研究纯化的GMFDGLSGV单体、二聚体、四聚体、八聚体重组蛋白刺激外周血单个核细胞(PBMC)产生的增殖反应和细胞毒活性,初步鉴定重组多聚体的免疫原性。结果10例HLA-A*0201阳性个体PBMC对八聚体的平均SI为37.4±3.07;增殖反应明显高于四聚体(平均SI为20.1±1.37)、二聚体(平均SI为14.1±1.06)、单体(平均SI为11.4±0.68)以及阴性对照(平均SI为1±0.10)引起的细胞增殖反应(P<0.01)。在抗原肽诱导的CTL细胞毒活性中,HLA-A*0201阳性个体PBMC对八聚体、四聚体、二聚体、单体及阴性对照组均表现不同程度的杀伤活性,平均活性分别为48.1±4.28、23.9±2.20、16.3±1.27、13.1±1.10、1.0±0.1(P<0.01)。结论本研究得到的重组优化八聚体具有较好的免疫原性,能有效刺激PBMC产生细胞增殖反应和细胞毒活性,为进一步开发有效的隐球菌疫苗打下了基础。
Objective To explore the ability of purified Cryptococcal antigen peptide GMFDGLSGV dimer, tetramer and octamer recombinant protein to stimulate the immune response of cytotoxic T lymphocyte (CTL), laying a foundation for the development of new Cryptococcus vaccine. Methods Proliferation and cytotoxic activity of purified GMFDGLSGV monomeric, dimeric, tetrameric and octameric recombinant proteins stimulated by peripheral blood mononuclear cells (PBMCs) were studied by cell proliferation and cytotoxicity assays. Preliminary identification of recombinant Multimeric immunogenicity. Results The average SI of octamer in 10 HLA-A * 0201-positive individuals was 37.4 ± 3.07; the proliferative response was significantly higher than that of tetramer (mean SI was 20.1 ± 1.37); dimers (average SI was 14.1 ± 1.06 ), Monomer (mean SI 11.4 ± 0.68), and negative control (mean SI 1 ± 0.10) (P <0.01). Among the antigenic peptide-induced CTL cytotoxic activity, PBMC of HLA-A * 0201-positive individuals showed different degrees of cytotoxic activity toward octamer, tetramer, dimer, monomer and negative control group, respectively 48.1 ± 4.28, 23.9 ± 2.20, 16.3 ± 1.27, 13.1 ± 1.10, 1.0 ± 0.1 (P <0.01). Conclusion The recombinant octamer obtained in this study has good immunogenicity, can effectively stimulate PBMC proliferation and cytotoxic activity, and lay the foundation for the further development of an effective Cryptococcus vaccine.