期刊文献+
任意字段
题名或关键词
题名
关键词
文摘
作者
第一作者
机构
刊名
分类号
参考文献
作者简介
基金资助
栏目信息

可溶性HLA-A*0201-PR1复合物的构建与鉴定 被引量：1

Construction and Characterization of Soluble HLA-A*0201-PR1 Complex
下载PDF
导出
摘要 为制备HLA-A*0201-PR1四聚体,本课题构建了可溶性的HLA-A*0201-PR1复合物。以原核表达的HLA-A*0201-BSP融合蛋白为重链,β2-微球蛋白(β2m)为轻链,与人工合成的HLA-A2限制性抗原肽PR1(蛋白酶3的第169-177氨基酸,VLQELNVTV)进行共折叠复性,以生成可溶性HLA-A*0201-PR1复合物。应用BirA酶对复性混和物进行生物素化,再经阴离子交换柱纯化,得到生物素化的可溶性HLA-A*0201-PR1复合物。应用凝胶过滤高效液相色谱分析可溶性HLA-A*0201-PR1复合物的生成率。利用HLA-A2构象特异性抗体(BB7.2)及链霉亲和素进行Western blot和ELISA分析,对生物素化的可溶性HLA-A*0201-PR1复合物进行鉴定。结果表明:在折叠体系中,主要含有HLA-A*0201-BSP聚合体、HLA-A*0201-PR1复合物及β2m,其中可溶性复合物生成率约18%。获得的HLA-A*0201-PR1复合物可在BirA酶作用下被有效生物素化。结论:成功地获得了生物素化的可溶性HLA-A*0201-PR1复合物,为进一步制备HLA-A*0201-PR1四聚体创立了基础。 This study was aimed to construct the soluble HLA-A ＊ 0201-PR1 complex for preparation of HLA-A ＊ 0201-PR1 tetramer. The recombinant HLA-A, 0201-BSP （BirA substrate peptide） fusion protein as heavy chain and 132-microglobulin （ 132 m） as light chain were expressed highly as insoluble aggregates in Escherichia coli and then purified with gel filtration, and the final purity reached above 90%. The two subunits were refolded to form an HLA-A ＊ 0201-peptide complex by dilution method in the presence of an antigenic peptide PR1, a HLA-A2-restricted peptide from proteinase 3 （ aa 169-177 ,VLQELNVTV）. Refolded HLA-A ＊ 0201-PR1 complex was biofinylated using a BirA enzyme and purified by anion exchange chromatography on a Q-Sepharose （ fast flow） column. The extent of re.constitution of the HLA-A ＊ 0201-PR1 complex was analyzed by HPLC gel filtration. The refolded and biotinylated products were detected by Western blot and ELISA with monoclonal antibody BB7.2 that recognized the natural conformations of HLA- A2 and streptavidin. The results showed that the refolded complex was composed of HLA-A＊ 0201-BSP aggregate, HLA-A ＊ 0201-PR1 complex and β2 m, and reconstitution yields of 18% with PR1 was obtained. Refolded HLA-A ＊ 0201-PR1 complex could be confirmed by practical immunological method and biotinylated efficiently. It is concluded that the refolding and biotinylation of HLA-A ＊ 0201-PR1 complex is successfully obtained. This work provides the basis for the preparation of HLA-A ＊ 0201-PR1 tetramer.
作者 孙万军 徐东刚 胡海兰 邹民吉 杜建芳 王金凤 蔡欣 王嘉玺 艾辉胜
机构地区 军事医学科学院附属医院血液科 军事医学科学院基础医学研究所 第二炮兵总医院血液科
出处 《中国实验血液学杂志》 CAS CSCD 2007年第2期352-356,共5页 Journal of Experimental Hematology
基金 军队"十一五"医药卫生科研基金资助项目 编号06MA316
关键词 HLA.抗原肽复合物 HLA-A＊0201-PRl复合物 HLA-A＊0201-PRl四聚体 HLA-antigenic oeptide complex： HLA-A ＊ 0201-PR1 complex HLA-A ＊ 0201-PR1 tetramer
分类号 Q78 [生物学—分子生物学] R733.7 [医药卫生—肿瘤]
  • 相关文献

参考文献13

  • 1Altman JD,Moss PA,Goulder PJ,et al.Phenotypic analysis of antigen-specific T lymphocytes.Science,1996; 274 (5284):94-96
  • 2Barnes E,Ward SM,Kasprowicz VO,et al.Ultrasensitive class Ⅰ tetramer analysis reveals previously undetectable populations of antiviral CD8+ T cells.Eur J Immunol,2004; 34:1570-1577
  • 3Palmowski M,Salio M,Dunbar RP,et al.The use of HLA class Ⅰ tetramers to design a vaccination strategy for melanoma patients.Immunol Rev,2002; 188:155-163
  • 4Yuan RR,Wong P,McDevitt MR,et al.Targeted deletion of T-cell clones using alpha-emitting suicide MHC tetramers.Blood,2004;104:2397-2402
  • 5孙万军,徐东刚,杜建芳,邹民吉,王金凤,蔡欣,王嘉玺,艾辉胜.人β_2-微球蛋白基因的克隆与原核中的表达[J].中国实验血液学杂志,2006,14(3):521-524. 被引量：1
  • 6孙万军,杜建芳,徐东刚,邹民吉,王金凤,蔡欣,王颖,王嘉玺,艾辉胜.HLA-A＊0201-BSP融合基因的构建与原核表达[J].中国实验血液学杂志,2006,14(5):976-980. 被引量：1
  • 7Garboczi DN,Hung DT,Wiley DC,et al.HLA-A2-peptide complexes:refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigen peptide.Proc Natl Acad Sci USA,1992; 89:3429-3433
  • 8何贤辉,徐丽慧,刘毅,蔡小嫦,曾耀英.加载HCMV抗原肽的HLA-A*0201单体及其四聚体制备和鉴定[J].生物工程学报,2004,20(3):382-388. 被引量：9
  • 9Dengler R,Munstermann U,al-Batran S,et al.Immunocytochemical and flow cytometric detection of proteinase 3 (myeloblastin) in normal and leukaemic myeloid cells.Br J Haematol,1995; 89:250-257
  • 10Molldrem JJ,Clave E,Jiang YZ,et al.Cytotoxic T lymphocytes specific for a nonpolymorphic proteinase 3 peptide preferentially inhibit chronic myeloid leukemia colony-forming units.Blood,1997;90:2529-2534

二级参考文献36

  • 1吴国光,邓志辉,高素青,程良红,金士正,周丹,李桢,邹红岩,张旋,魏天莉,程曦,王大明.6965名汉族骨髓供者HLA多态性分析[J].中华血液学杂志,2004,25(8):473-477. 被引量：96
  • 2萨姆布鲁克J 弗里奇EF 曼尼阿蒂斯T.分子克隆实验指南.第3版[M].北京:科学出版社,2002.1595.
  • 3Harty JT, Tvinnereim AR, White DW. CD8 + T cell effector mechanisms in resistance to infection. Annu Rev Immunol, 2000, 18:275- 308
  • 4Meidenbauer N, Hoffmann TK, Donnenbergc AD. Direct visualization of antigen-specific T cells using peptide-MHC-class Ⅰ tetrameric complexes. Methods, 2003, 31:160- 171
  • 5Appay V, Rowland-Jones SL. The assessment of antigen-specific CD8 + T cells through the combination of MHC class Ⅰ tetramer and intracellular staining. J Immunol Methods, 2002, 268:9 - 19
  • 6Altman JD, Moss P AH, Goulder PJR et al. Phenotypic analysis of antigen-specific T lymphocytes [ published erratum appears in Science 1998, 280 ( 5371 ): 1821 ]. Science, 1996, 274: 94 - 96
  • 7Garboczi DN, Hung DT, Wiley DC. HLA-A2-peptide complexes:Refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci USA, 1992, 89: 3429- 3433
  • 8Bousso P. Generation of MHC-peptide tetramers: a new opportunity for dissecting T-cell immune responses. Microbes Infec, 2000, 2:425 - 429
  • 9Wills MR, Carmichael AJ, Mynard K et al. The human cytotoxic Tlymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity and T-cell receptor usage of pp65-specific CTL. J Virol, 1996, 70: 7569-7579
  • 10Gratama JW, van Esser JWJ, Lamers CHJ et al. Tetramer-based quantification of cytomegalovirus ( CMV)-specific CD8 + T lymphocytes in T-cell depleted stem cell grafts and after transplantation may identify patients at risk for progressive CMV infection. Bl

共引文献8

同被引文献9

  • 1Halverson DC, Schwartz GN, Carter C, et al. In vitro generation of allospecific human CD8^+ T cells of Tcl and To2 phenotype. Blood, 1997;90(5) :2089 -2096.
  • 2Rezvani K, Yong AS, Savani BN, et al. Graft-versus-leukemia effects associated with detectable Wilms tumor-1 specific T lymphocytes after allogeneic stem-cell transplantation for acute lymphoblastic leukemia. Blood, 2007; 110(6) :1924 -1932.
  • 3Hui-Sheng Ai, Chun-Hua Zhao, Mei Guo, et al. Mixed hematopoietic chimerism-a new strategy for preventing GVHD in nonmyeloablative allogeneic stem cell transplantation. Experimental Hematology, 2002 ;30 (5) : 119.
  • 4Asemissen AM, Keilholz U, Tenzer S, et al. Identification of a highly immunogenic HLA-A^* 01-Binding T cell epitope of WT1. Clin Cancer Res, 2006 ; 12 (24) : 7476 - 7482.
  • 5Weiden PL, Flournoy N, Thomas ED, et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneicmarrow grafts. N Engl J Med ,1979 ; 300(19) : 1068 - 1073.
  • 6Schulenburg A, Fischer M, Kalhs P, et al. Immune recovery after conventional and non-myeloablative allogeneic stem cell transplantation. Leuk Lymphoma, 2005 ; 46 ( 12 ) : 1755 - 1760.
  • 7Tobery TW, Dubey SA, Anderson K, et al. A comparison of standard immunogenicity assays for monitoring HIV type 1 gagspecific T cell responses in Ad5 HIV Type 1 gag vaccinated human subjects. AIDS Res Hum Relax)viruses, 2006 ;22 ( 11 ) : 1081 - 1090.
  • 8Molldrem 22, Lee PP, Wang C, et al. A PR1-Human leukocyte antigen-A2 tetramer can be used to isolate low-frequency cytotoxic T lymphocytes from healthy donors that selectively lyse chronic myelogenous leukemia. Cancer Res, 1999 ; 59 ( 11 ) :2675 - 2681.
  • 9Baker J, Vemeris MR, Ito M, et al. Expansion of cytolytic CD8 ( + ) natural killer T cells with limited capacity for graft-versushost disease induction due to interferon gamma production. Blood, 2001 ; 97(10) :2923 -2931.

引证文献1

中国实验血液学杂志
2007年 第2期

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部