药学学报, 2012, 47(10): 1317-1322
引用本文:
周雅琼, 张 娟, 金海珍, 何 远, 王 彤, 王 旻. 抗EGFR/抗KDR双特异性单链抗体的构建及表达[J]. 药学学报, 2012, 47(10): 1317-1322.
ZHOU Ya-qiong, ZHANG Juan, JIN Hai-zhen, HE Yuan, WANG Tong, WANG Min. Construction and expression of an anti-EGFR/anti-KDR bispecific single-chain diabody[J]. Acta Pharmaceutica Sinica, 2012, 47(10): 1317-1322.

抗EGFR/抗KDR双特异性单链抗体的构建及表达
周雅琼, 张 娟*, 金海珍, 何 远, 王 彤, 王 旻*
(中国药科大学, 天然药物活性物质与功能国家重点实验室, 江苏 南京 210009)
摘要:

针对EGFRVEGFR信号通路的相互作用导致的耐药性, 为增强抗EGFR或抗VEGFR2抗体单独使用疗效不佳的癌症的治疗效果, 本文利用重叠PCR将抗人EGFR胞外区单链抗体基因scFv-E10和抗人VEGFR2 (KDR) 的单链抗体基因scFv-AK404R融合在一起, 得到抗EGFR/KDR双特异性单链抗体 (bispecific single- chain diabody, scDb) 基因, scDb的连接肽序列为15个氨基酸 (G4S)3, 在肽链的C端引入His标签及myc标签。将scDb基因连接入载体pHEN2, 转化大肠杆菌HB2151并诱导表达, 经镍柱纯化获得电泳纯scDb蛋白, 产量约为570 μg·L−1发酵液。ELISA测定显示, scDbEGFR胞外区的结合与亲本scFv-E10相当, KDR胞外区的结合略低于亲本scFv-AK404R, 表明抗EGFR/KDR scDb可与EGFRKDR两种抗原特异性结合, 可用于进一步的抗肿瘤活性研究。

关键词:   
Construction and expression of an anti-EGFR/anti-KDR bispecific single-chain diabody
Abstract:

Bispecific antibodies have been exploited as both cancer immunodiagnostics and cancer therapeutics, which have shown promises in clinical trials in cancer imaging and therapy.  To improve the anti-tumor effect, an scDb (bispecific single-chain diabody) was constructed from the variable domain genes of two scFvs (single-chain variable fragment antibodies) directed against human EGFR (epidermal growth factor receptor) and VEGFR2 (vascular endothelial growth factor receptor 2) extracellular domains.  The anti-EGFR/ anti-KDR scDb was constructed into pHEN2 plasmid and expressed in Escherichia coli HB2151 host.  After purification by one-step affinity chromatography of IMAC, scDb protein was characterized by Western blotting.  The yield of scDb protein was 570 μg per liter medium. scDb bound to EGFR as efficiently as the parental antibody scFv-E10, while a little bit weaker than the parental antibody scFv-AK404R when bound to KDR.  In conclusion, the scDb protein could bind both EGFR and KDR specifically and could be applied for further anti-tumor research.

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