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共价连接β2m的HLA-A2/IgG1二聚体的构建、表达与纯化

Stabilization of the Divalent HLA-A2/IgG1 Molecule during Acidical Elution by Covalent Association of β2m with HLA-A2/IgG1 Molecule
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摘要 目的HLA-A2/IgG1二聚体经蛋白A(SPA)亲合层析柱浓缩纯化时容易发生β2 m解离,为了解决这个问题,研究构建与表达β2 m-HLA-A2/IgG1融合蛋白。方法利用基因重组技术构建β2 m-HLA-A2/IgG1融合基因真核表达载体pcDNA3.1(+)-[β2 m-HLA-A2/IgG1],将其转染721.221细胞系,筛选高表达β2 m-HLA-A2/IgG1融合蛋白的细胞株;收集细胞培养上清,SPA亲合层析柱浓缩纯化,检测该蛋白的纯化效率。结果β2 m-HLA-A2/IgG1融合基因转染721.221细胞后可表达正确构象的β2 m-HLA-A2/IgG1二聚体,Western blot显示β2 m-HLA-A2/IgG1融合蛋白的分子量与预期大小一致;经SPA亲合层析柱纯化后,β2 m-HLA-A2/IgG1融合蛋白较HLA-A2/IgG1融合蛋白的纯化效率约高5倍。结论共价连接β2 m的HLA-A2二聚体有助于HLA-A2二聚体在低pH值的环境下保持构象完整。 Objective To prepare a more stable divalent β2m-HLA-A2/IgG1 molecule by covalent association of β2m with the HLA-A2 heavy chain, which is expected to be more stable during the purification by staptylococcal protein A （SPA） affinity chromatography. Methods The chimeric gene expressing divalent β2m-HLA-A2/IgG1 molecule was constructed by fusing β2m with the extracellular domains of HLA-A2 and the CH1-Hinge-CH2-CH3 domains of human IgG1 heavy chain. The molecule was expressed in transfected 721. 221 cells, and purified by SPA affinity chromatography. Sandwich ELISA and Western blot by specific antibody were performed to identify the fusion protein to check its intact conformation. Results Molecular weight and conformation of the divalent β2m-HLA-A2/IgG1 molecule expressed in transfected 721. 221 cells were the same as the non-covalent counterpart. The purification efficiency of the divalent β2m-HLA-A2/IgG1 molecule with SPA was about 7 times higher than that of the non-covalent counterpart. Conclusion The divalent β2m-HLA-A2/IgG1 molecule covalently associated with β2m shows more stability, which is able to keep intact conformation at low pH circumstance during purification with SPA column.
作者 郝娟 段红霞 李庆 孙伟 钟茂华 翁秀芳 吴雄文
机构地区 华中科技大学同济医学院基础医学院免疫学系
出处 《华中科技大学学报（医学版）》 CAS CSCD 北大核心 2009年第1期1-5,共5页 Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金 国家自然科学基金资助项目(No.30772040)
关键词 β2m-HLA—A2/IgG1融合蛋白 真核表达 亲合层析 构象完整 divalent β2m-HLA-A2/IgG1 molecule eukaryotic expression affinity chromatography intact conformation
分类号 R34-33 [医药卫生—基础医学]
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参考文献9

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华中科技大学学报（医学版）
2009年 第1期

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