卵巢癌抗独特型单链抗体的人源化——抗独特型微抗体的构建与表达

为了降低人抗鼠抗体反应 ,获得满意的免疫原性 ,将模拟人卵巢癌抗原的抗独特型单链抗体人源化 .采用重叠PCR和基因工程的技术 ,将 6B11ScFv基因的轻链和重链颠倒 ,成为 6B11VL VH.再与人IgG1铰链区和CH3区的基因进行融合 (VL VH CH3) ,构建抗独特型微抗体的原核表达载体 .转化E .coliBL2 1(DE3)后用IPTG诱导表达 .经SDS PAGE分离显示 ,在 5 0kD左右处有一诱导蛋白带 .不连续非变性凝胶电泳显示 ,表达产物分子量为 10 0kD左右 .采用ELISA、竞争抑制实验、West ern印迹对其进行活性测定 .结果表明 ,人源化的抗独特型微抗体具有特异性双价结合卵巢癌单克隆抗体COC16 6 9和识别人免疫球蛋白IgG1的活性 ,成功地将鼠源的scFv的人源化

Humanization of Ovarian Carcinoma Anti-idiotype Single-chain (V_L-V_H-CH3) Antibody

In order to reduce the human anti mouse antibody response and obtain optimal antigenicity, anti idiotype single chain which mimicking ovarian cancer antigen has been humanized. Using overlap PCR and DNA recombinant technique, the sequence of 6B11 ScFv was changed by heavy chain and light chain reversal. Prokaryotic expression vector was produced by genetic fusion of 6B11V L V H to human IgG1 hinge and CH3 region. Transformed E.coli BL21(DE3) was propagated and induced by IPTG. SDS PAGE showed that a protein band with molecular weight of 50 kD appeared as the expected size after transformation. Molecular weight of 100 kD might be examined by electrophoresis in non denaturing systems. The fusion protein was analyzed with ELISA, inhibition ELISA tests and Western blotting, respectively. The humanized anti idiotype minibody showed the capacity of bivalent binding to ovarian cancer monoclonal antibody coc166 9 and goat anti human immunoglobulin IgG1. It is a useful reagent for clinical uses.