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目的:纯化可刺激人γδ+ T细胞增殖的结核杆菌耐热多肽抗 原。方法:采用快速蛋白液相色谱仪(FPLC)S-100分子筛层析柱,对结核杆菌耐热抗原(Mtb-Ag)初步分离。将获得的结核杆菌耐热Mr低的多肽抗原(Mtb-LW-Ag)洗脱峰,分别再经FPLC Mono Q离子交换层析柱进一步纯化,并且采用流式细胞仪对获得的Mtb-LW-Ag进行刺激γδ+ T细胞增殖活性的测定。结果:Mtb-Ag经FPLC S-100柱可分离出1个大分子蛋白峰A和3个M,低的多肽峰(B,C,D)。Mr低的多肽峰分别经Mono Q柱层析,鉴定出B峰含有6个主峰,C峰含有1个主峰,D峰含有8个主峰。对Mtb-LW-Ag及其纯化多肽进行活性检测,发现多肽峰B和C以及纯化多肽B-Ⅲ和C-主肽均可显著刺激γδ+ T细胞扩增。结论:利用FPLC法可快速高效地从Mtb-Ag中纯化出多种Mr低的多肽,而且其中的B-Ⅲ多肽和C-主肽可能是促进γδ+ T细胞活化增殖的主要多肽。
OBJECTIVE: To purify Mycobacterium tuberculosis heat-resistant polypeptide antigen that can stimulate the proliferation of human γδ + T cells. Methods: Mtb-Ag of Mycobacterium tuberculosis was initially separated by FPLC S-100 molecular sieve column. The obtained Mtb-LW-Ag peptide was further purified by FPLC Mono Q ion exchange chromatography, and the obtained Mtb-LW-Ag fragment was purified by flow cytometry. Ag to measure γδ + T cell proliferative activity. Results: One macromolecular protein peak A and three M, low polypeptide peaks (B, C, D) were isolated from Mtb-Ag by FPLC S-100 column. Mr low peptide peaks were Mono Q column chromatography, identified B peak contains six main peaks, C peak contains a main peak, D peak contains eight main peak. The activity of Mtb-LW-Ag and its purified polypeptide was detected, and it was found that peptide peaks B and C and purified polypeptide B-III and C-a-peptide all significantly stimulated γδ + T cell expansion. CONCLUSION: A variety of Mr low peptides can be rapidly and efficiently purified from Mtb-Ag by FPLC method. And the B-Ⅲ polypeptide and C-peptide may be the main peptides that promote the activation and proliferation of γδ + T cells.