结核特异性CD4^+ α/β TCR四聚体细胞株筛选技术的建立和应用  

Establishment and application of cell strains to screen specific CD4^+ α/β TCR tetramers

作　　者：姚亚男[1] 黄宇虹[1] 王娟[1] 陈伊[1] 任亮亮[1] 赖小敏[1] 

机构地区：[1]中山大学中山医学院微生物学教研室/热带病防治研究教育部重点实验室/海洋微生物功能分子广东省高校重点实验室/广东省重大传染病预防和控制技术中心,广州510080

出　　处：《中国医药生物技术》2014年第2期88-94,共7页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金面上项目(81271779);"十二五"国家科技重大专项传染病防治专项(2012ZX10004903-004-002)

摘　　要：目的应用不同的MTB多肽以及不同的HLA-DR基因构建的膜表面表达结核抗原肽/HLA-DR复合物的S2恒定细胞株,即人工APC筛选、鉴定结核特异性CD4+α/βTCR四聚体。方法以PE标记的CD4+α/βTCR四聚体、FITC标记的抗HLA-DR抗体(L243),分别与未诱导表达或CuSO4诱导表达后的膜表面表达结核抗原肽/HLA-DR的果蝇S2恒定细胞株,以及与无多肽或结合有结核多肽的、表达HLA-DR的S2细胞株共孵育,流式细胞技术检测分析TCR四聚体与各种细胞株结合率的差异。结果 TCR四聚体6M、6N均与2#细胞株,即C5/HLA-DRB1*0404(1.73%、5.93%)等具有较高的阳性结合率;结核抗原肽孵育后,一些人工APC的四聚体阳性结合率有所提高;所筛选的9个TCR四聚体均与2#细胞株有较高的结合率。结论膜表达结核抗原肽/HLA-DR的S2细胞株,可以应用于结核特异性CD4+α/βTCR四聚体的筛选、鉴定;构建的9个CD4+α/βTCR四聚体均为结核特异性。Objective To screen and identify MTB antigen-specific CD4＋ α/β TCR tetramers using different membrane expressing MTB antigen peptide/HLA-DR complex cell strains （artificial APCs）. Methods PE labeled CD4＋α/β TCR tetramers and FITC labeled anti-HLA-DR antibody （L243） were incubated with membrane expressing MTB antigen peptide/HLA-DR cell strains （before and after induction） or S2 cell strains expressing only HLA-DR or HLA-DR binding with MTB peptides E6, E7, C5, C14 or tumor peptide respectively, and then different binding capacity of TCR tetramers were detect and analyzed by flow cytometry. Results Both TCR tetramers 6M and 6N showed a high positive rate with No. 2 cell strain （C5/HLA-DRB1＊0404, 1.73% and 5.93%respectively）. After incubation with MTB peptide, the percentage of tetramer positive rate increased in some of the artificial APCs, and high affinity was observed between all of the nine TCR tetramers and No. 2 cell strain respectively. 〈br〉 Conclusion Membrane expressing MTB antigen peptide/HLA-DR complex cell strains could be used to screen and identify MTB-specific CD4＋α/βTCR tetramers, and all of the nine tested TCR tetramers are MTB-specific.

关 键 词：结核多肽 人工抗原递呈细胞 CD4+T 细胞 TCR四聚体 

分 类 号：R392[医药卫生—免疫学]