抗肿瘤血管三结构域单链抗体ＶＨ／Ｌ的构建与表达

以本室研制的一株抗肿瘤血管单克隆体ＡＡ９８为基础，采用ＰＣＲ扩增抗体ＡＡ９８基因的重链可变区（ＶＨ）和轻链（Ｌ），以重链恒定区１（ＣＨ１）５′端１２个氨基酸的序列作为连接肽，并将连接肽中的Ｌys变为Ｓer,构建ＶＨ－连接肽－Ｌ三结构域单链抗体。重组ＶＨ／Ｌ单链抗体在大肠杆菌中得到了高效表达，其表达量占菌体总蛋白质的２０％。，表达的蛋白质在菌内形成包含体，经凝胶过滤法复性，获得了有抗原结合活性的ＶＨ／Ｌ。该三结构域单链抗体的成功构建和复性，为重组抗体片段的研制提供了借鉴。

A Three-domain Antibody Fragment VH/L Specific to Tumor Blood Vessels

AA98 is a specificaally anti-angiogenic antibody generated in our lab. The heavy chain variable region (VH) attached with mutagenized 36 nucleotides sequence derived from the heavy chain constant region1 (CH1) was amplified VH and light chain (L) were inserted into high-level expression vector pET21a successively, thus pET21a-VH/L was constructed. VH/L was expressed in E. coli BL21 (DE3) after induction with IPTG. The expression of VH/L was 20% of the total bacterial proteins. The refolding of VH/L was conducted by dilution and gel filtration chromatography. The refolded VH/L could bind to HUVEC specifically. Its affinity to the antigen is similar to that of recombinant AA98Fab, but lower than that of the parent antibody AA98.