| 张 番,苏 金,王婷婷,李惠晨,边 寰,卜 歆.DDR2结构域与人IgG Fc融合载体的构建、表达与蛋白纯化[J].现代生物医学进展英文版,2017,17(34):6653-6656. |
| DDR2结构域与人IgG Fc融合载体的构建、表达与蛋白纯化 |
| Construction, Expression and Protein Purification of DDR2 Domain and Human IgG Fc Fusion Vector |
| Received:August 20, 2017 Revised:September 10, 2017 |
| DOI：10.13241/j.cnki.pmb.2017.34.010 |
| 中文关键词: 人IgG Fc 分子克隆 分泌型蛋白 |
| 英文关键词: Human IgG Fc Clone Secretory protein |
| 基金项目:陕西省自然科学基础研究计划项目-面上项目（2016JM8045） |
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| 中文摘要: |
| 摘要 目的：构建DDR2与人IgG Fc融合的分泌型真核表达载体，验证其表达，纯化具有生物活性的分泌型蛋白。方法：分析DDR2蛋白质结构功能域，设计载体构建策略；扩增DDR2胞外结构域片段，将其克隆至PsecTag2B载体(带有人IgG Fc标签)中，在293T细胞验证其表达；用Protein G纯化柱纯化蛋白。结果：DDR2胞外结构域DS/Fc融合载体测序正确，转染293T细胞中后可成功分泌到细胞上清中，进一步用亲和层析法纯化得到高浓度蛋白。结论：重组载体特异性分泌DDR2胞外区蛋白，引入的Link序列是稳定分泌蛋白构象的基础，融合了人IgG Fc序列，可以作为DDR2配体拮抗剂靶向抑制DDR2与其天然配体的相互作用，对临床研究有重要作用。 |
| 英文摘要: |
| ABSTRACT Objective: To construct a secreted eukaryotic fusion vector of DDR2 and human IgG Fc and verify the expression of fusion protein and finally to obtain a biologically active secretory protein. Methods: First, we analyzed the structure of DDR2 protein and designed a method for vector construction. The fragment of DDR2 extracellular domain was amplified by PCR and cloned into PsecTag2B vector with human IgG Fc tag. Then the expression of the recombinant vector was verified in 293T cells. The fusion protein was purified by Protein G affinity column. Results: The DS/Fc vector was correctly sequenced. The fusion protein was successfully harvested from 293T cell culture medium, which was further purified and amplified by affinity column. Conclusion: The recombination vector specifically secreted the protein of DDR2 extracellular domain, which introduced a link sequence in order to stabilize the protein conformation. The fusion protein could act as an antagonist of DDR2 ligands inhibiting the binding of DDR2 to its specific collagen ligand, which contributed to the progress of DDR2 research. |
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