| 齐洁琼,高 红,庄婷婷,于文功,顾玉超.单链抗体scFv-GCN4在大肠杆菌中的重组表达及活性检测[J].现代生物医学进展英文版,2017,17(4):606-609. |
| 单链抗体scFv-GCN4在大肠杆菌中的重组表达及活性检测 |
| Expression of Recombinant Antibody scFv-GCN4 in Escherichia coli and Determination of its Biological Activity |
| Received:March 27, 2016 Revised:April 25, 2016 |
| DOI：10.13241/j.cnki.pmb.2017.04.002 |
| 中文关键词: 单链抗体 scFv-GCN4 重组表达 大肠杆菌 活性检测 |
| 英文关键词: Single-chain variable fragment scFv-GCN4 Recombinant expression Escherichia coli Activity detection |
| 基金项目:国家自然科学基金项目（81272264） |
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| 中文摘要: |
| 摘要 目的：重组表达融合GST或MBP标签的单链抗体scFv-GCN4，对其进行分离纯化，并检测其生物学活性。方法：构建pBAD-MBP-scFv-GCN4及pBAD-GST-scFv-GCN4表达载体，使用大肠杆菌(Escherichia coli) Top10菌株表达并亲和纯化重组蛋白MBP-scFv-GCN4及GST-scFv-GCN4。构建pET30a-Nus-GCN4载体，使用E. coli BL21(DE3)菌株表达重组蛋白Nus-GCN4及Nus。以重组的GCN4为特异性抗原，通过Pull-down 技术和Western Blot技术，检测MBP-scFv-GCN4及GST-scFv-GCN4的抗体活性及特异性。结果：重组菌株E. coli Top10可高效表达可溶性的MBP-scFv-GCN4和GST-scFv-GCN4蛋白，通过亲和纯化，均得到了高纯度的重组蛋白。重组菌株E. coli BL21(DE3)可高效表达可溶性的Nus与Nus-GCN4蛋白。Pull-down及Western Blot结果显示，重组蛋白MBP-scFv-GCN4及GST-scFv-GCN4均可以高效、特异地识别重组的GCN4抗原。结论：重组抗体GST-scFv-GCN4及MBP-scFv-GCN4均可在E. coli中高效表达，并且具有良好的抗体活性及特异性。 |
| 英文摘要: |
| ABSTRACT Objective: To express and purify the single chain Fv antibodies against GCN4 (scFv-GCN4) which fused with GST-Tag or MBP-Tag, and to determine the characteristics of the recombinant antibody. Methods: Construct recombinant plasmids pBAD-MBP-scFv-GCN4 and pBAD-GST-scFv-GCN4, purify recombinant antibodies expressed in E. coli Top10. Construct recombinant plasmids pET30a-Nus-GCN4, express recombinant proteins Nus and Nus-GCN4 in E. coli BL21(DE3). Identify the biological activity and specificity of the recombinant antibodies by Pull-down and Western Blot. Results: Recombinant antibodies MBP-scFv-GCN4 and GST-scFv-GCN4 could be expressed and purified efficiently. Nus and Nus-GCN4 could be expressed efficiently in E. coli BL21(DE3). The methods of Pull-down and Western Blot proved that recombinant antibodies could recognize GCN4 specifically and efficiently. Conclusion: Recombinant antibodies MBP-scFv-GCN4 and GST-scFv-GCN4 could be expressed efficiently in E. coli, and possessed high biological activity and specificity to GCN4. |
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