为增强抗EGFR或抗VEGFR2(KDR)单克隆抗体的抗肿瘤作用,作者将抗人EGFR单克隆抗体(Cetuximab)可变区基因和抗人KDR单克隆抗体(m Ab-04)可变区基因融合并与人Ig G1的Fc段基因通过柔性肽连接在一起,得到全人源Ig G样抗EGFR/抗KDR双特异性抗体(Bi-Ab)的基因。将Bi-Ab基因插入载体p PICZα,转化毕赤酵母X-33并诱导表达。发酵液经硫酸铵沉淀法初步提取后采用Protein A亲和层析柱进一步分离纯化,得到电泳纯的Bi-Ab蛋白,其产量约为2.3 mg/L发酵液。表面等离子共振法(SPR)分析显示,Bi-Ab与EGFR或KDR的结合亲和力与其亲本Cetuximab或m Ab-04相近,表明该双特异性抗体Bi-Ab能与EGFR和KDR特异性结合,可用于进一步的抗肿瘤活性研究。 To enhance the anti-tumor effect of anti-EGFR or anti-VEGFR2 (KDR) monoclonal antibodies, the authors transformed the Cetuximab variable region gene and the anti-human KDR monoclonal antibody (m Ab-04) Fused and fused to the Fc-segment gene of human Ig G1 by a flexible peptide to obtain a gene of a full-human Ig G-like anti-EGFR / anti-KDR bispecific antibody (Bi-Ab). The Bi-Ab gene was inserted into vector p PICZα, transformed into Pichia pastoris X-33 and induced for expression. The fermented liquid was initially extracted by ammonium sulfate precipitation and further purified by Protein A affinity chromatography to obtain pure Bi-Ab protein. The yield was about 2.3 mg / L of fermentation broth. Surface plasmon resonance (SPR) analysis showed that the binding affinity of Bi-Ab to EGFR or KDR was similar to that of its parent Cetuximab or m Ab-04, indicating that the bispecific antibody Bi-Ab specifically binds EGFR and KDR and can be used in Further antitumor activity studies.