表皮生长因子受体胞外区在哺乳动物细胞HEK293T中的分泌表达和鉴定

目的:融合表达表皮生长因子受体(EGFR)胞外功能区,为制备针对该分子的特异性抗体提供可用的靶标抗原。方法:通过PCR扩增EGFR胞外区基因,将其克隆入真核表达载体pABhFc中,重组质粒瞬时转染HEK293T细胞,进行EGFR的瞬时分泌表达,纯化获得电泳纯级的分泌蛋白,并通过ELISA、Western印迹、Biacore3000系统对融合蛋白进行鉴定。结果:经测序证实扩增得到了正确的EGFR胞外区基因序列,SDS-PAGE初步确认获得了单、双体的EGFR胞外区,ELISA检测证实双体融合蛋白hFc-EGFR可与商业化的EGF特异性结合,Western印迹检测证实单体融合蛋白His-EGFR可与商业化抗体特异性结合;经Biacore3000蛋白分子相互作用系统测定,双体融合蛋白hFc-EGFR与商业化抗体爱必妥的亲和力达0.5 nmol/L。结论:利用哺乳动物细胞HEK293T分泌表达系统获得了结构正确的单、双体2种类型的EGFR胞外区融合蛋白纯品,将用于抗EGFR特异性抗体的筛选。

Secretion Expression of Extracellular Region of EGFR Using Mam malian Cell and its Identification

Objective: To obtain epidermal growth factor receptor(EGFR) extracellular region,which will be used as the target antigens for selection anti-EGFR specific antibody.Methods: EGFR extracellular region gene was amplified by PCR and cloned into the eukaryotic expression vector pABhFc,and the recombinant plasmids were transfected transiently into HEK293T cells.The recombinant proteins were expressed and secreted,then they were purified respectively.The purified protein were identified by ELISA,Western blot and Biacore3000 system.Results: The gene sequence of EGFR extracellular domain was confirmed by DNA sequencing,then the result of SDS-PAGE exhibited that the monomer and dimer EGFR extracellular domain successfully expressed.ELISA dem onstrated the specific binding of dimer recombinant hFc-EGFR and commercial EGF,and Western blot proved the specific binding of monomer recombinant His-EGFR and the commercial specific antibody.Affinity of commercial antibody Erbitux and dimer recombinant hFc-EGFR achieved to 0.5 nmol/L calculated by the Biacore3000 protein interactions system.Conclusion: Monomer and dimer two different recombinant EGFR extracellular domain with correct structure were obtained by using mammalian cell expression system,which found a good basis for the selec tion of anti-EGFR specific antibodies.