单链抗体scFv-GCN4在大肠杆菌中的重组表达及活性检测

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p ET30a-Nus-GCN4载体,使用E.coliBL21(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WesternBlot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.coliBL21(DE3)可高效表达可溶性的Nus与Nus-GCN4蛋白。Pull-down及WesternBlot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。

Expression of Recombinant Antibody scFv-GCN4 in Escherichia coli and Determination of its Biological Activity

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.