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加载白血病抗原肽PR1的HLA-A＊0201四聚体制备 被引量：1

Preimrafion of HLA-A ＊ 0201 tetramer loaded with PRI antigen peptide
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摘要 目的制备加载白血病抗原肽PR1的HLA-A＊0201四聚体。方法构建HLA-A＊0201-BSP和β2-微球蛋白（β2M）原核表达载体，进行目的蛋白表达、纯化。在体外将PR1（VLQELNVIV）与纯化的HLA-A＊0201-BSP、β2M通过稀释复性折叠成HLA-A＊0201-PR1复合物。利用BirA酶进行生物素标记。再经阴离子交换纯化得到生物素化的HLA-A＊0201-PR1复合物单体。此单体与PE标记的链霉亲和素按4：1比例混合，即可形成四聚体。结果成功制备了HLA-A＊0201-PRl四聚体。结论制备的HLA-A＊0201-PR1四聚体为定量检测白血病患者体内PR1抗原特异性的细胞毒性T淋巴细胞提供了有力工具。 Objective To construct PR1-specific HIA-A ＊ 0201-peptide tetramer, and to identify PRl-specific cytotoxic T lymphocytes（CTL） in patients with leukemia.Methods Recombinant HIA-A ＊ 0201 heavy chain and β2-micmglobulin （β2M） were prepared in Escherichia coli cells transformed with pBV220 and pET-17b vectors, respectively. Only the extracellular region HLA-A ＊ 0201 was cloned by RT-PCR from HLA-A2 donor,and a 15-amino acid BirA substmte peptide （BSP） for BirA-dependent biotinylation was added into the COOH-terminus of HIA-A ＊ 0201 heavy chain.HLA-A ＊ 0201-BSP and β2 M were refolded to form a HLA-A ＊ 0201-peptide complex by dilution method in the presence of a leukemia-associated antigen peptide PR1, a HLA-A ＊ 0201 restricted peptide from proteinase 3（169-177 AA, VLQELNVTV）.Refolded HLA-A ＊ 0201-PR1 complex monomer was biofinylated using a BirA enzyme and purified by anion exchange chromatography on a Q-Sepharose （fast flow）column. Tetramers were generated by mixing biotinylated HIA-A ＊ 0201-PR1 monomer with streptavidin - PE at a molar ratio of 4：1.Retmlts The expression levels of pBV220-HIA-A ＊ 0201-BSP and pET-β2 M were 28% and 32% of total bacterial proteins estimated from SDS-PAGE,respectively. They were mainly expressed in the form of inclusion body and final purity was 90%. The refolded HLA-A ＊ 0201-PR1 complex monomer was detected by Westem blot with monoclonal antibody BB7.2 that recognized the natural conformations of HIA-A2. HIA-A ＊ 0201-PR1 tetramer was successfully obtained. Conclusion High-efficient expressions of HIA-A ＊ 0201-BSP and β2 M recombinant protein provide the basis for the preparation of HIA-A ＊ 0201 tetramers to study the function of CTL. Especially, using HLA-A ＊ 0201-PR1 tetranmr can detect the frequencies of the PR1-specific CD8 ＊ CTL directly in vitro.
作者 孙万军 艾辉胜 徐东刚 邹民吉 杜建芳 王金凤 蔡欣 王嘉玺
机构地区 军事医学科学院附属医院血液科 北京第二炮兵总医院血液科 军事医学科学院基础医学研究所
出处 《中华微生物学和免疫学杂志》 CAS CSCD 北大核心 2007年第4期293-298,共6页 Chinese Journal of Microbiology and Immunology
基金 军队“十一五”医药卫生科研基金（编号06MA316）
关键词 人类白细胞抗原 四聚体技术 白血病相关抗原 抗原特异性细胞毒性T淋巴细胞 Human leukocyte antigen Tetramer technology Leukemia-associated antigen Antigen-specificcytotoxic T lymphocyte
分类号 R686 [医药卫生—骨科学]
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参考文献11

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二级参考文献8

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中华微生物学和免疫学杂志
2007年 第4期

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