中国医学科学院学报2013，Vol.35Issue(3)：281-285,5.DOI:10.3881/j.issn.1000-503X.2013.03.008
含末端半胱氨酸人类表皮生长因子受体2亲和体的重组表达与亲和纯化
Expression and Affinity Purification of Recombinant Human Epidermal Growth Factor Receptor-2 Affibody with C-terminal Cystein
蔡炯 1郑堃 1党永红 1李方1
作者信息
- 1. 中国医学科学院 北京协和医学院 北京协和医院核医学科,北京100730
- 折叠
摘要
Abstract
Objective To prepare the modified ZHER2∶V2 affibody with amino-terminal HEHEHE sequence and carboxyl-terminal GGGC sequence by gene recombinant expression,which is the basis for invasive HER2 imaging with affibody.Methods The encoded affibody gene was optimized by codon preference of E.coli with gene designer software.The N-terminal of affibody was fused with HEHEHE sequence,while the C-terminal was fused with GGGC sequence.The synthetic gene was confirmed by Hind Ⅲ endonuclease restriction and gene sequencing.The human epidermal growth factor receptor-2 (HER2) affibody gene was subcloned into pET22b (+) plasmid and transformed into competent BL21 (DE3) bacteria.The expression of modified affibody was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) and identified by SDS-PAGE.The affibody was purified by nickel affinity binding and imidazole elution.The purified affibody was labeled with 68Ga and its affinity was determined by saturation analysis with HER2-positive cells MDA-MB-361.Results The affibody gene containing N-terminal HEHEHE and C-terminal GGGC sequences were confirmed by Hind Ⅲ endonuclease restriction and gene sequencing.A newly expressed 8 × 103 protein was expressed from the induced recombinant bacteria identified by SDS-PAGE after sub-cloning HER2 affibody gene into pET22b (+) plasmid,transforming recombinant plasmid into competent BL21 (DE3) bacteria and inducing the recombinant bacteria with IPTG.The expressed protein was purified from nickel agarose by 60 mmol/L imidazole eluting.The affinity Kd value of 68Ga labeled affibody to HER2 positive MDA-MB-361 cells was 1.5 nmol/L.Conclusion The affiibody ZHER2∶V2 containing N-terminal HEHEHE and C-terminal GGGC was successfully prepared by gene optimization,recombinant expression and affinity purification.关键词
人类表皮生长因子受体2/亲和体/表达/纯化Key words
human epidermal growth factor receptor-2/affibody/expression/purification分类
生物科学引用本文复制引用
蔡炯,郑堃,党永红,李方..含末端半胱氨酸人类表皮生长因子受体2亲和体的重组表达与亲和纯化[J].中国医学科学院学报,2013,35(3):281-285,5.
