%0 Journal Article
%T Preparation of H-2Db Tetramer and Its Application in Enumerating the CD8+ T Cells Specific for Lymphocytic Choriomeningitis Virus<br>H-2Db四聚体的制备及其在检测LCMV特异性CD8+ T细胞中的应用
%A Yi Liu
%A Lihui Xu
%A Xuesi Zeng
%A Jianfang Sun
%A Xianhui He
%A <br>刘 毅
%A 徐丽慧
%A 曾学思
%A 孙建方
%A 何贤辉
%J 生物工程学报
%D 2008
%I 
%X 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-2Db-restricted CD8+ T cells in immunotherapy, we prepared the H-2Db tetramer and verified its effectiveness in enumerating the CD8+ T cells specific for the lymphocytic choriomeningitis virus (LCMV). First, the cDNA encoding H-2Db heavy chain was cloned by RT-PCR from the spleen of a C57BL/6 mouse. The expression vector for H-2Db-BSP, i.e. the ectodomain of H-2Db fused to a BirA substrate peptide (BSP), was constructed and overexpressed in E. coli BL21(DE3). Then, the denatured H-2Db-BSP was refolded in the presence of human beta2-microglobulin as well as the GP33-41 peptide (KAVYNFATC, KAV) of LCMV. The biotinylated H-2Db/KAV molecules were purified, then bound to streptavidin-PE and tetramerized. Finally, the prepared H-2Db 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 (< or = 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-2Db tetramer will facilitate the study of the immune responses of antigen-specific CD8+ T cells in the experimental immunotherapy on the mice with H-2Db allele background.
%K H-2Db
%K MHC tetramer
%K lymphocytic choriomeningitis virus
%K prokaryotic expression<br>H-2Db
%K MHC四聚体
%K 淋巴细胞性脉络丛脑膜炎病毒
%K 原核表达
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=8F8B032D34FF2F3729DB7EBD5315AFA2&yid=67289AFF6305E306&vid=B91E8C6D6FE990DB&iid=0B39A22176CE99FB&sid=B4E8EA49DAAEB84F&eid=03E56C113B4E5A88&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=0&reference_num=17