摘要
目的:我们曾从噬菌体抗体库中克隆到一株VH第3骨架区中含有异常序列的抗HBsAg人Fab基因,本工作拟探讨该VH基因中异常序列对抗体活性的影响.方法:采用重叠PCR方法去除这段异常序列,构建表达载体,转化大肠杆菌表达出抗HBsAg噬菌体抗体和可溶性Fab,测定了它们与抗原的结合活性.结果:与原抗体比较,改造后的抗体与抗原的结合活性明显下降,分子模建提示这段异常序列对VH CDR_1和CDR_2的部分氨基酸残基的位置有一些影响,测定改造前Fab的亲和力约为0.55×10~9M^(-1),改造后Fab因抗原结合活性过低,无法检测其亲和力.结论:提示这段异常序列在体内就可能存在于该VH之中.
Objective: Previously we cloned a human anti-HBsAg Fab. the VH of which contained a stretch of extra-sequence. In this work we investigated the effect of the extra-sequence on the activity of the anti-HBsAg Fab. Methods: The extra-sequence was removed by overlap PCR. The modified Fab was expressed in E. coli, and its Ag binding activity was tested and compared with the original Fab. Results: The modified Fab showed drastically decreased Ag binding activity. Molecular modeling demonstrated changes in some residues in VH CDR1 and CDR2. The associate constant of the original Fab is about 0. 55×109M-1, while the ELISA signal of the modified Fab was too weak to determine the affinity. Conclusion: The results suggest that the VH containing extra-sequence may exist in vivo.
出处
《转化医学杂志》
2000年第1期1-5,共5页
Translational Medicine Journal
基金
本工作受国家科委863项目(Z18-01-02-03)资助
