H-2D^b四聚体的制备及其在检测LCMV特异性CD8^＋T细胞中的应用  

Preparation of H-2D^b Tetramer and Its Application in Enumerating the CD8^+ T Cells Specific for Lymphocytic Choriomeningitis Virus

作　　者：刘毅[1] 徐丽慧[2] 曾学思[1] 孙建方[1] 何贤辉[3] 

机构地区：[1]中国医学科学院中国协和医科大学皮肤病研究所,南京210042 [2]暨南大学生命科学技术学院生物工程研究所,广州510632 [3]暨南大学生命科学技术学院组织移植与免疫实验中心,广州510632

出　　处：《生物工程学报》2008年第2期278-284,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金项目(Nos．30672474，30572199，30230350);~~中国医学科学院中国协和医科大学皮肤病研究所科研基金~~

摘　　要：MHC四聚体技术是研究抗原特异性淋巴细胞应答的关键技术之一。为研究H-2D^b基因型(如C57BL/6)小鼠的特异性CD8^+T细胞免疫应答,需要建立H-2D^b四聚体制备技术平台。首先以RT-PCR方法克隆H-2D^b重链基因的cDNA,进而构建H-2D^b胞外域与生物素化酶BirA底物肽(BSP)融合蛋白的表达载体,并在大肠杆菌中获得表达。在LCMV GP_(33-41)抗原肽(KAVYNFATC,KAV)和人β_2-微球蛋白存在时,通过稀释法复性获得H-2D^b/KAV单体。该单体经生物素化并纯化后与PE-链亲和素按4:1的比例混合,即形成四聚体。通过流式细胞术检测经KAV肽免疫的C57BL/6小鼠体内的LCMV特异性CD8^+T细胞的频率.结果表明在外周血、引流淋巴结和脾脏中均可检测到一定频率的LCMV特异性CD8^+T细胞,其中以对外周血标本染色的效果最佳。成功建立了小鼠H-2D^b四聚体制备技术平台,为监测及分析基于H-2D^b基因型小鼠的实验性免疫治疗创造了条件。Major histocompatibility complex （MHC） tetramer technology offers a powerful means to study specific T cell populations of interest. To investigate the immune response of H-2D^b-restricted CD8^＋ T cells in immunotherapy, we prepared the H-2D^b tetramer and verified its effectiveness in enumerating the CD8^＋ T cells specific for the lymphocytic choriomeningitis virus （LCMV）. First, the cDNA encoding H-2D^b heavy chain was cloned by RT-PCR from the spleen of a C57BL/6 mouse. The expression vector for H-2D^b-BSE i.e. the ectodomain of H-2D^b fused to a BirA substrate peptide （BSP）, was constructed and overexpressed in E. coil BL21（DE3）. Then, the denatured H-2D^b-BSP was refolded in the presence of human β2-microglobulin as well as the GP33-41 peptide （KAVYNFATC, KAV）of LCMV. The biotinylated H-2D^b/KAV molecules were purified, then bound to streptavidin-PE and tetramerized. Finally, the prepared H-2D^b KAV tetramer reagent was verified by detecting the CD8^＋ T cells specific for HCMV in KAV peptide vaccinated C57BL/6 mouse, with a mouse receiving subcutaneous injection of only adjuvant as negative control. The results showed that the tetramer positive rates were 0.27%, 0.11%, and 0.24% within the CD8^＋ T cell populations in the peripheral blood, draining lymph nodes, and spleen of vaccinated mouse, respectively. There was only very low background staining （≤0.01%） of those samples from the control mouse. Beside, the best results were achieved in the staining of the peripheral blood sample. In conclusion, the established procedure of preparing H-2D^b tetramer will facilitate the study of the immune responses of antigen-specific CD8^＋ T cells in the experimental immunotherapy on the mice with H-2D^b allele background.

关 键 词：H-2D^b MHC四聚体 淋巴细胞性脉络丛脑膜炎病毒 原核表达 

分 类 号：R392[医药卫生—免疫学]