抗HBs人V_H基因中异常序列对抗体活性影响的研究

A stretch of extra-sequence in an anti-HBs V_H gene is indispensable for the antibody activity

目的 从噬菌体抗体库中克隆到 1株VH 第 3骨架区中含有异常序列的抗HBs人Fab基因 ,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方法去除了这段异常序列 ,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VHCDR1和CDR2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 5 5×10 9mol/L ,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可能存在于该VH 之中。

Objective Previously we cloned a human anti-HBs Fab, the V H 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-HBs Fab. Methods Following removal of the extra-sequence by overlap PCR, the modified Fab gene was expressed in E.coli , and its Ag binding activity 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 V H CDR1 and CDR2. The associate constant of the original Fab is about 0.55×10 9mol/L, while the ELISA signal of the modified Fab was too weak to determine the affinity. Conclusion The V H containing extra-sequence may exist in vivo.