摘要目的 构建及应用结核分枝杆菌(Mycobacterium tuberculosis,Mtb)特异性CD4+α/βT细胞受体(TCR)四聚体筛选细胞系.方法 从Mtb多肽阳件反应结核患者中,PCR扩增HLA-DR β链,构建HLA-DR β链和加载有结核抗原肽的HLA-DR α链全长基因的表达载体pMT-HLA-DRB及pMT-HLA-DRA-P,磷酸钙转染法转入果蝇S2(Schneider2)细胞中进行表达配对,细胞免疫组化法初步鉴定表达情况,并应用所建立细胞系以流式细胞术筛选Mtb特异性CD4+α/βTCR四聚体.结果 细胞免疫组化法及流式细胞术鉴定显示获得细胞膜上表达结核抗原肽/HLA-DR复合物的稳定S2细胞株,并筛选出2种Mtb特异性CD4+α/β TCR四聚体.结论 成功构建Mtb特异性CD4+α/β TCR四聚体筛选细胞系,为分析鉴定Mtb特异反应性TCR四聚体,探索开发结核诊断新方法 及新型结核疫苗的研制提供了工具.

abstractsObjective To construct and apply a cell line screening Mycobacterium tuberculosis (Mtb)-specific tetramers of CD4+α/β T cell receptor(TCR). Methods The β chains of HLA class Ⅱ (DR) were amplified from tuberculosis patients by PCR. The pMT-HLA-DRB expression vectors that carries the HLA-DR 13 chain and pMT-HLA-DRA-P expression vectors which carries the genes of HLA-DR α chain loaded with Mtb antigen were transfected into S2 cells with the method of calcium phosphate transfection. The expressed Mtb peptide/HLA-DR complexes were primarily identified by the method of cell immunohistochemistry. The cell lines expressing Mtb peptide/HLA-DR complexes were used to screen tetramers of CD4+ TCR by flow cytometry. Results S2 cell lines expressing Mtb peptide/HLA-DR complexes on the cell surface were obtained, two kinds of Mtb specific tetramers of CD4+α/β TCR were screened. Conclusion S2 cell lines expressing Mtb peptide/HLA-DR complexes on the cell surface provide the solid basis of the further research on the TCR tetramers and are helpful for exploring new diagnostic study methods about tuberculosis and developing new vaccines.