抗表皮生长因子受体的单链抗体ER(Fv)的构建及其抗肿瘤活性研究

Construction of a Single-chain Fv Antibody against Epidermal Growth Factor Receptor and Its Antitumor Activity

目的利用大肠杆菌表达抗表皮生长因子受体(EGFR)的单链抗体并对其抗肿瘤活性进行研究。方法构建含有抗EGFR单链抗体基因片段的重组质粒,IPTG诱导大肠杆菌表达单链抗体ER(Fv),用Ni离子亲和柱纯化单链抗体。ELISA测定ER(Fv)与纯EGFR抗原的结合能力;流式细胞术分析ER(Fv)与肿瘤细胞的结合;MTT法检测ER(Fv)对肿瘤细胞体外增殖的影响;动物试验测定ER(Fv)的体内抑瘤效果。结果应用基因工程菌表达带有组氨酸标签肽(His-tag)的单链抗体ER(Fv),相对分子质量约为27×103,主要以包涵体形式存在,收获量约为10 mg.L-1。ELISA测定ER(Fv)与纯EGFR抗原的亲和常数为4.0×107 L.mol-1。单链抗体能与肿瘤细胞表面的EGFR发生特异性结合,其与3种EGFR高表达肿瘤细胞的解离常数皆为10-7 mol.L-1。细胞增殖实验显示,单链抗体可抑制EGFR高表达肿瘤细胞的增殖。体内抑瘤实验表明,单链抗体对人鳞状上皮癌A431裸鼠移植瘤具有中度的生长抑制作用,5,10和20 mg.kg-1 3个剂量组35 d的抑瘤率分别为43.9%、46.7%和54.8%,与对照组相比存在显著差异。结论成功地表达了抗EGFR单链抗体ER(Fv),其对肿瘤细胞具有良好的亲和力和一定的生长抑制作用,为研制靶向EGFR的抗体药物提供基础。

OBJECTIVE To express single-chain Fv（scFv） antibody against epidermal growth factor receptor（EGFR） with Escherichia coli,and study its antitumor activity.METHODS Recombinant plasmids containing scFv gene fragment were constructed and transformed into competent E.coli BL21（DE3）starTM.The scFv antibody,ER（Fv）,was produced after IPTG induction,and then purified with Ni ion affinity chromatography.ELISA was used for measuring the binding efficiency of ER（Fv） to EGFR,and flow cytometry was applied to assay the binding activity between ER（Fv-LDP） and tumor cells.The cytotoxicity of ER（Fv） against tumor cells in vitro was assessed by MTT assay,and the tumor inhibitory effect in vivo was observed in nude mice bearing human epidermoid carcinoma A431 xenograft.RESULTS ER（Fv） with His-tag was produced in the form of inclusion bodies,and was purified with Ni ion affinity chromatography.Finally 10 mg of purified protein were obtained from 1 L of culture medium.ER（Fv） possessed powerful binding affinity for EGFR with an affinity constant（Ka）of 4.0×107 L·mol-1.ER（Fv） protein bound specifically to the membrane of EGFR-overexpressing tumor cells with an dissociation constant（Kd） of 10-7 mol·L-1.ER（Fv） showed potent cytotoxicity against tumor cells.In A431 xenografts,ER（Fv） presented antitumor efficacy.The tumor growth inhibition rates by ER（Fv） at dose of 5,10 mg·kg-1,and 20 mg·kg-1 were 43.9%,46.7% and 54.8%,respectively.CONCLUSION The preparation of ER（Fv） with EGFR binding affinity and antitumor activity provides the basis for the development of EGFR-targeted antibody-based drugs.