期刊文献+

小鼠H-2D^b-BSP融合基因的构建与表达

Construction and Expression of Mouse H-2D^b-BSP Fusion Gene
下载PDF
导出
摘要 目的:构建小鼠H-2Db-BSP融合基因,并诱导其在大肠杆菌中表达.方法:采用RT-PCR方法从C57BL/6小鼠淋巴细胞中扩增出H-2Db链的胞外段基因,经双酶切置换已构建的HLA-A*0201-BSP重组体中的HLA-A*0201胞外段序列,使H-2Db与BirA酶底物肽(BSP)序列融合,构建H-2Db-BSP融合基因表达载体,转化大肠杆菌BL21(DE3)菌株,筛选重组子,并经IPTG诱导融合蛋白表达.结果:构建的H-2Db-BSP融合基因插入正确且序列与GenBank一致;经1mmol/L浓度的IPTG诱导后4h蛋白表达达到高峰,且融合蛋白以包涵体形式存在.结论:成功构建H-2Db-BSP融合基因,并诱导其在大肠杆菌得以表达,为进一步构建H-2Db四聚体,研究T细胞应答提供了物质基础. Objective:To construct the fusion gene of mouse H-2Db-BSP, and investigate its expression in E.coli. Methods:The H-2Db was amplified by RT-PCR method from total RNA of leukomonocyte which isolated from C57BL/6 (B6) mouse (H-2b). The sequence of H-2Db was used to replace the HLA-A*0201 sequence in a pre-existing HLA-A*0201-BSP vector, to generate an H-2Db-BSP expressing vector. After transformation of E.coli BL21 ( DE3) cells with the vectors, the expression of fusion protein was induced by isopropyl -D-thiogalactoside (IPTG). Results: The sequence of the H-2Db-BSP was matched with that of Genbank. Upon induction with 1mmol/ L IPTG 4 hours, the fusion protein was expressed in E.coli BL21 cells, and large amounts of recombinant protein accumulated in intracellular inclusion bodies. Conclusion:We have successfully constructed the fusion gene of mouse H-2Db-BSP, and confirmed that the fusion gene can be expressed in E.coli BL21 cells. High efficient expression of H-2Db-BSP fusion protein lays the foundation for the constructing H-2Db tetramers to explore corresponding of T cell recognition.
出处 《江汉大学学报(自然科学版)》 2010年第3期95-97,101,共4页 Journal of Jianghan University:Natural Science Edition
基金 武汉市科技局科技计划项目(201051099415-07)
关键词 克隆 H-2Db 生物素化序列 cloning H-2Db BirA substrate peptide
  • 相关文献

参考文献9

  • 1Whitelegg A M, Oosten L E, Jordan S, et al. Investigation of peptide involvement in T cell alloreeognition using recombinant HLA class I multimers [J]. J Immunol, 2005, 175(3): 1706-1714.
  • 2Scharnagl N C, Klade C S. Experimental discovery of T- cell epitopes: combining the best of classical and contem- porary approaches [J]. Expert Rev Vaccines, 2007, 6(4): 605-615.
  • 3Altman J D, Moss P A, Goulder P J R, et al. Phenotypic analysis of antigen-specific T lymphocytes [J]. Science, 1996, 274(5284) : 94-96.
  • 4Novak E J, Liu A W, Gebe J A, et al . Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4 + T cell epitopes from complex antigens [J]. J Immunol, 2001, 166(11) : 6665-6670.
  • 5Meidenbauer N, Hoffmann T K, Donnenbergc A D. Direct visualization of antigen-specific T cells using peptide- MHC-class I tetrameric complexes [J]. Methods, 2003, 31 (2): 160-171.
  • 6Giannoni F, Barnett J, Bi K, et al. Clustering of T cell ligands on artificial APC membranes influences T cell activation and protein kinase C theta translocation to the T cell plasma membrane[J]. J Immunol,2005,174 (6):3204-3211.
  • 7翁秀芳,陈浩,吴雄文,梁智辉,韩军艳,黄亚非,龚非力.可溶性HLA-A2-肽复合物的体外折叠与生物素化[J].细胞与分子免疫学杂志,2004,20(3):265-268. 被引量:10
  • 8卢小玲,吴雄文,翁秀芳,梁智辉,李青,龚非力.sHLA-A*2402融合蛋白的制备与特性研究[J].现代免疫学,2005,25(2):137-140. 被引量:3
  • 9Guillaume P, Dojcinovic D, Luescher I E Soluble MHC- peptide complexes: tools for the monitoring of T cell responses in clinical trials and basic research [J]. Cancer Immun, 2009, 9:7.

二级参考文献19

  • 1Maziarz RT, Fraser J, Srominger JL, et al. The human HLAspecific monoclonal antibody W6/32 recognizes a discontinuous eptiope within the alpha 2 domain of murine H-2Db [J].Immunogenetics, 1986,24(3):206-208.
  • 2Yuriko S. Improved generation of HLA class I/peptide tetramers[J]. J Immunol Methods, 2002.271(1-2):177-184.
  • 3.[EB].http://www.emory.edu/WHSC/lETRAMER/protocol.html,.
  • 4Parker KC, Silver ML, Wiley DC, An HLA-A-A2-/β2m/peptide complex assembled from subunits expressed separately in E. coli[J]. Mol Immunol, 1992,29(3):371-378.
  • 5Altman JD, Moss PAH, Goulder PJR, et al. Phenotypic analysis of antigen-specific T lymphocytes[J]. Science, 1996,274(5284):94-96.
  • 6Ee LT, Paige LJ, Matthew FM. Activation of antigen-specific T cells by artificial cell constructs having immobilized multimeric peptede-class Ⅰ complexes and recombinant B7-Fc proteins[J]. J Immunol Methods, 2001,249(1-2):111-119.
  • 7Yasunari N, Shuichi K, Hisao T, et al. Analysis of the CD8-positive T cell response in Japanese patients with chronic hepatitis using HLA-A* 2402 peptide tetramers [J]. J Med Virol,2003,70(1):51-61.
  • 8Schatz PJ. Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli[J]. Biotechnology(NY),1993,11(10):1138-1143.
  • 9Kiyotaka K, Naomi H, Ayumi K, et al. Tetramer-assisted identification and characterization of epitopes recognized by HLA-A* 2402-restricted Epstein-Barr virus-specific CD8+ T cells[J]. Blood, 2003,101(4):1460-1467.
  • 10Garboczi DN, Hung DT, Wiley DC, et al. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides[J]. Proc Natl Acad Sci USA, 1992, 89(8): 3429-3433.

共引文献10

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