采用RT-PCR方法,利用第一骨架区通用引物扩增重链Fd段和κ链的基因,克隆到Fab表达载体中,在大肠杆菌中获得了表达但未检测到抗原结合活性.根据已克隆的3H11 VL、VH的真实序列,重新设计κ链及Fd段5′端引物,分别将骨架区引物在κ链及Fd段5′端所造成的氨基酸残基改变纠正为原始序列,构建分别含有矫正后κ链或矫正后Fd以及二个链均得到矫正的Fab表达载体,这些载体在大肠杆菌中均获得类似水平的表达,对任何一个链的矫正均可部分恢复Fab段胃癌细胞的结合活性.结果说明在构建小分子抗体时,PCR引物引入的轻、重链可变区氨基端氨基酸残基的改变可严重影响所表达抗体的抗原结合活性.
The cDNAs of κ chain and Fd fragment of anti-gastric cancer mAb 3H11 were amplified by RT-PCR using degenerate primers for framework region 1(FR1) and cloned into an Fab expression vector. Expression of Fab could be detected but with no antigen binding activity. Then the Vκ and Fd genes were corrected to its genuine sequence by PCR mediated mutagenesis. The reconstructed Fab containing either corrected κ chain or Fd or both were expressed in E.coli at similar level. Correction of any one of the V region genes could partially resume the antigen binding activity. This result indicated that PCR primers introduced Vκ and Fd N terminal changes may seriously affect the antigen binding activity.
李竞,王琰,王卓智,刘群英,化冰,陈宇萍,朱迎春,董志伟.抗胃癌单抗3H11可变区氨基端序列对抗体活性的影响[J].生物化学与生物物理进展,1999,26(4):369-373
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