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Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli
Piao, WH; Song, XG; Liu, MC; He, Y; Zhang, HH; Xu, WX; Li, ZL; Zhang, HQ; Ling, SG; Wang, GQ
关键词Hla-a0201-bsp And Beta-2m Genes Pbv220 Vector Expression Purification
刊名PROTEIN EXPRESSION AND PURIFICATION
2004-06-01
DOI10.1016/j.pep.2004.01.002
35期:2页:210-217
收录类别SCI
文章类型Article
WOS标题词Science & Technology
类目[WOS]Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
研究领域[WOS]Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
关键词[WOS]LIMITING DILUTION ANALYSIS ; CELLULAR IMMUNE-RESPONSES ; CD8(+) T-CELLS ; MELANOMA PATIENTS ; FLOW-CYTOMETRY ; ELISPOT ASSAY ; FREQUENCIES ; LYMPHOCYTES ; TETRAMERS ; REEVALUATION
英文摘要

Tetramer analysis is a novel technique in immunological research that has dramatically changed our knowledge of the immune response to pathogens, tumors and autoimmune disease. Through the formation of major histocompatibility complex (MHC)-peptide tetrameric complexes, it can provide accurate counts of antigen-specific T-cells and it allows their phenotypical and functional analysis. The tetramer is composed of the human leukocyte antigen (HLA) heavy chain, beta-2 microglobulin (beta-2m), the nominal peptide, and streptavidin. The HLA heavy chain and the beta-2m are expressed in Escherichia coli. But up to now, all laboratories have been expressing these two proteins by using isopropyl P-D-thiogalactopyranoside IPTG [1-6]. IPTG is very expensive, and it is tedious and laborious to induce expression protein. So it is difficult to scale up to express the objective protein [7]. To address this problem, extracellular fractions of HLA-A0201 and beta-2m (absent signal peptide) genes were cloned from peripheral blood mononuclear cells (PBMCs) by RT-PCR. DNA coding for a Gly-Ser linker and a BSP (15-amino acid substrate peptide for BirA-dependent biotinylation) was added to the COOH-terminus of the extracellular fraction of HLA-A0201 by PCR, using an HLA-A0201 as the template. Then the HLA-A0201-BSP and beta-2m genes were cloned into pBV220 vector and expressed, respectively. The expressed proteins were purified and detected by ELISA and Western blot analyses. High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs. (C) 2004 Elsevier Inc. All rights reserved.

语种英语
WOS记录号WOS:000221626300006
引用统计
被引频次:4[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/53755
专题北京大学第一临床医学院
作者单位1.Peking Univ, Hosp 1, Beijing 100034, Peoples R China
2.Yanbian Univ, Coll Med, Jilin 133000, Peoples R China
3.Acad Mil Med Sci, Inst Basic Med Sci, Beijing 100850, Peoples R China
推荐引用方式
GB/T 7714
Piao, WH,Song, XG,Liu, MC,et al. Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli[J]. PROTEIN EXPRESSION AND PURIFICATION,2004,35(2):210-217.
APA Piao, WH.,Song, XG.,Liu, MC.,He, Y.,Zhang, HH.,...&Wang, GQ.(2004).Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli.PROTEIN EXPRESSION AND PURIFICATION,35(2),210-217.
MLA Piao, WH,et al."Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli".PROTEIN EXPRESSION AND PURIFICATION 35.2(2004):210-217.
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