伴侣素包含T复合蛋白1ε亚基(CCT5)是与人γδT细胞相关的自身抗原

Chaperonin containing T-complex protein 1 subunit epsilon (CCT5) serves as γδ T cell-related autoantigen

目的探讨CCT5与γδT细胞及自身免疫病的关系。方法 PCR扩增人CCT5的基因,表达并纯化CCT5重组蛋白;用3条CCT5蛋白的肽段免疫小鼠,制备并筛选抗CCT5的单克隆抗体;固相化CCT5重组蛋白,体外扩增人外周血单个核细胞中的γδT细胞;用制备的单抗通过ELISA检测正常人、类风湿性关节炎(RA)及系统性红斑狼疮(SLE)患者血浆中CCT5蛋白的含量;用流式细胞技术分析不同亚型γδT细胞的比例,并与血浆CCT5含量做相关性分析。结果 PCR扩增获得CCT5基因,目的片段长度为1 750 bp;经SDS-PAGE分析可见重组CCT5蛋白分子质量约为65 ku,并获得了纯化。通过筛选,获得两株抗CCT5单克隆抗体对。重组CCT5蛋白能在体外扩增外周血γδT细胞;而血浆CCT5浓度与Vγ9和Vδ2 T细胞的比例呈显著正相关。在RA及SLE患者血浆中,可溶性CCT5的浓度显著低于正常对照(P<0.05)。结论 CCT5是与人Vγ9δ2γδT细胞识别的相关自身抗原,有助于我们深入了解Vγ9δ2γδT细胞在自身免疫病中的作用。

Objective To examine the relationship between CCT5,γδ T cell and autoimmune diseases. Methods Recombinant CCT5 protein was cloned,expressed and purified in E. coli. Three peptides of CCT5 protein were used to prepare for anti-CCT5 monoclonal antibodies. Purified CCT5 protein was used to expand γδ T cells from peripheral blood mononuclear cells（ PBMC）. Plasma level of CCT5 in healthy donors,patients with rheumatoid arthritis（ RA） and systemic lupus erythematosus（ SLE） were detected by ELISA assays. The correlation analysis between plasma CCT5 concentration and the percentage of different subtypes of γδ T cells measured by flow cytometry was made.Results The CCT5 gene was amplified by PCR and the length of the target fragment was 1 750 bp. The expressed65 ku CCT5 protein was purified and validated by SDS-PAGE. Two paired monoclonal anti-CCT5 antibodies werescreened to detect CCT5 protein in plasma. Immobilized recombinant CCT5 protein was able to induce specific significant amplification of peripheral γδ T cells. Correlation analysis of 10 healthy donors indicated significant correlation between the plasma CCT5 concentration and the proportion of Vγ9 and Vδ2 γδ T cells. The plasma CCT5 concentration significantly decreased in autoimmune diseases patients,including RA and SLE. Conclusions These data suggest that CCT5 could be a novel Vγ9δ2 γδ T cell-related factor in autoimmune diseases,which deepen the understanding of Vγ9δ2 γδ T cell function in autoimmune diseases.