目的:利用噬菌体抗体库和导向选择技术，以抗HAb18G嵌合抗体轻链为模板，筛选人源性抗肝癌抗体Fd片段。方法: 利用RT-PCR自肝癌患者外周血淋巴细胞中扩增全套人抗体重链Fd基因片段，克隆入含嵌合L链的展示载体pComb3X，建立人－鼠杂合Fab噬菌体抗体库。然后，利用大肠杆菌表达的非融合HAb18GE为抗原进行亲和筛选，利用pⅢ融合抗体的形式进行克隆鉴定，并对筛选出的杂合抗体进行初步的功能检测。结果:构建成功库容量为2×10 7 PFU的杂合人 鼠噬菌体抗体库，利用非融合表达的HAb18GE进行6轮筛选。ELISA及流式细胞仪检测，其中7个克隆呈特异性阳性反应。其中克隆AP6-2和AP6-7亲和力较高，测序显示其基因序列相同，且与亲本抗体恒定区序列相同，属IgG2亚类，可变区属VH3家族。结论:通过本实验，利用嵌合轻链为模板，进行Fd片段替换，成功筛选到人源性抗体Fd片段。

Objective: To select anti-HAb18G (hepatoma associated antigen) human Fd fragments with guided selection of L chain of chimeric Fab-HAb18.Methods:The human Fd repertories genes were amplified by RT-PCR from PBMC of hepatoma patients, and cloned into the vector pComb3X with chimeric L chain to construct the human-mouse hybrid Fab phage library. HAb18GE, extracellular region of HAb18G, was used as antigen to screen. The positive clones was obtained by ELISA and FCM with pⅢ-fusion Fab antibody. The DNA sequences were analyzed. Results: A human-mouse Fab antibody library were constructed with 2×10 7 PFU. After 6 rounds panning, 7 positive clones were obtained with ELISA and FCM. And sequences of 2 clones with better affinity were same. The CH1 belonged to the IgG2 class as the parent Fd, and the variable region belonged to VH3 family. Conclusions:Through the construction of the HuMFab phage antibody library and chemeric L chain-guided selection, we get the available human Fd fragments for subsequent research.

国家自然科学基金资助项目（编号30070842）