| 白乌仁图雅 赵亚璁 朱燕锋 石 宇 孙 剑.sTACI-Fc-Myc 重组质粒的构建、原核表达及活性鉴定[J].现代生物医学进展英文版,2015,15(2):217-220. |
| sTACI-Fc-Myc 重组质粒的构建、原核表达及活性鉴定 |
| Construction, Expression and Activity Analysis of sTACI-Fc-Myc Protein |
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| DOI： |
| 中文关键词: B 细胞活化因 子 TACI-Fc-Myc 融合蛋白 原核表达 |
| 英文关键词: B-cell activating factor sTACI-Fc-myc fusion protein Prokaryotic expression |
| 基金项目:国家自 然科学基金项目（ 81273308） |
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| 中文摘要: |
| 目 的： 构 建 sTACI-Fc-Myc 重组质粒， 并进行原 核表达和纯 化具有生物活性的 融合蛋白 。 方法： 通过 PCR 法获得
sTACI-Fc-Myc 重组片段， 然后把融合基因 片 段与原核载体 pET28a 连接在一起， 并构建 pET28a－ sTACI－ Fc－ Myc 重组子， 并转
入 BL21 (DE3)中进行表达，用 蛋白 A 凝胶亲和层析柱进行纯化及酶联免疫吸附剂（ ELISA)法测定其生物学活性。 结果： 获得了
sTACI-Fc-Myc 重组质粒， 且该质粒可以在 BL21 (DE3)中表达， 亲和层析柱纯化后纯度可达到 95 %以上，与 BAFF 的结合活性具
有剂量依赖性， 浓度达到 5 ng/uL 时，两者的吸附达到 饱和。 结论： 成功构建了 sTACI－ Fc－ Myc 原核表达载体， 并使有生物学活
性的融合蛋白 在 BL21 (DE3)上获得了稳定表达， 为进一步研究并筛选高活性 BAFF 拮抗肽奠定了 基础。 |
| 英文摘要: |
| Objective:To construct recombinant plasmid of sTACI-Fc-myc gene and to induce its prokaryotic express and to
purify the protein.Methods:sTACI-Fc-myc gene was obtained by PCR amplification and then combined with pET28a to construct
pET28a-sTACI-Fc-myc recombinant plasmid. The recombinant plamid was transformed into BL21 (DE3), and then
sTACI-Fc-myc protein was expressed and purified by protein A affinity column chromatography. The purity and activity of
sTACI-Fc-myc were identified by ELISA, SDS-PAGE, respectively.Results:The sTACI-Fc-myc gene was obtained and the recombinant
plasmid can express in BL21 (DE3). The purity can be as high as 95% after affinity chromatography. The biological activity of the
purified sTACI-Fc-myc protein was analyzed. sTACI-Fc-myc could bind BAFF specifically in a dose-dependent manner, and the
saturated concentration was 5 ng/uL.Conclusion:The fusion vector is constructed successfully and a purified recombinated
sTACI-Fc-myc protein is obtained, which lays a foundation for further studies of BAFF antagonist peptibody. |
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