抗人P185^(erbB2)的scFv-Fc融合蛋白的表达及免疫功能分析  

Expression and Characterization of Single-chain Fv-Fc Fusion Protein against Human P185^(erbB2)

作　　者：解志刚[1] 郭宁[1] 施明[1] 冯建男[1] 于鸣[1] 孙瑛勋[1] 沈倍奋[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850

出　　处：《生物化学与生物物理学报》2003年第4期371-374,共4页

基　　金：国家高技术研究发展计划 (863计划 )资助项目 (No .2 0 0 1AA2 15 0 81)~~

摘　　要：为提高鼠源单抗用于体内治疗的效应功能及降低人抗鼠抗体反应 ,将编码抗人P185 erbB2 单抗轻、重链可变区 (VL、VH)融合构建的单链抗体 (scFv)基因片段与人IgG1的Fc区基因片段融合构建了scFv Fc融合基因 ,并将其克隆到哺乳动物细胞表达载体pCIDN中。用重组载体转染CHO细胞 ,通过G4 18筛选获得稳定高表达克隆。更换无血清培养基培养 ,经重组蛋白质A亲和层析柱纯化scFv Fc融合蛋白。融合蛋白质在还原SDS PAGE中表现为5 2kD的条带 ;与SK BR 3细胞裂解液共培育可特异性地沉淀出P185 erbB2 蛋白 ;FACS分析表明融合蛋白质识别P185 erbB2 蛋白的胞外段 ;ELISA测定融合蛋白质对细胞表面抗原P185 erbB2 的亲和常数为 7.5× 10 -10 (mol/L) -1。构建scFv Fc融合基因 ,将其克隆到表达载体 ,进一步稳定表达抗P185 erbB2 的scFv Fc融合蛋白 ,为进一步的体外、体内研究鼠源单抗治疗效果创造了条件。In order to increase therapeutic effects and decrease immunogenicity of mouse McAb, the single-chain Fv (scFv) created by fusing the light and heavy chain variable region genes of anti-human P185 erbB2 McAb was conjugated to the Fc gene of human IgG1 to construct a scFv-Fc fusion gene. The scFv-Fc fusion gene was cloned into the expression vector pCIDN. The scFv-Fc fusion protein was synthesized as secreted two-chain molecules in CHO cells, and purified by affinity chromatography on recombinant protein A. A special 185 kD P185 erbB2 protein was immunoprecipitated by the scFv-Fc fusion protein. The fluorescence-activated cell sorting (FACS) using SK-BR-3 cells as the target indicated that the fusion protein could bind to the extracellular domain of P185 erbB2 . The affinity of the scFv-Fc fusion protein, determined by ELISA, was K= 7.5×10 -10 (mol/L) -1 . This work laid basis for further studies on the anti-P185 erbB2 scFv-Fc fusion protein.

关 键 词：P185^erbB2 单链抗体-Fc融合蛋白 肿瘤 鼠源单抗 免疫功能 免疫疗法 

分 类 号：Q786[生物学—分子生物学]