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目的 :在嵌合HAb18 Fab抗体的基础上 ,利用载体pComb3X ,采用导向选择链更替技术建立全人源性Fab基因库。方法 :用RT PCR自肝癌患者外周血单个核细胞 (PBMC)中 ,扩增全套人抗体Fd基因片段和L链基因。首先 ,将Fd基因克隆入已含嵌合L链基因的展示载体pComb3X/CL中 ,建立人 -鼠杂合的Fab基因库 ,以原核表达的非融合HAb18G的胞外区片段为抗原 ,筛选杂合的Fab基因 ,以得到人Fd基因。然后应用筛选出的Fd基因与人L链基因库配对 ,建立全为人Fab的基因库 ,并筛选全人Fab基因。将IPTG诱导的表达菌裂解 ,取其上清对pⅢ Fab融合蛋白的功能进行分析 ,并对其编码基因进行测序。结果 :建立了库容为 2× 10 7的杂合Fab基因库 ,经 6轮筛选后得到 7株杂合Fab基因。利用筛选的人Fd基因建立了库容为 0 .8× 10 7的全人Fab基因库 ,经 4轮筛选得到 2株亲和力较高、特异性强的人Fab基因。测序结果显示 ,2株Fab基因具有相同的Fd基因序列 ,与亲本抗体同属IgG2亚类 ;L链基因为κ型 ,可变区均属于Vκ3家族。结论 :利用导向选择链更替技术 ,筛选出特异性较强、完全人源化的抗肝癌抗原HAb18G的Fab抗体 ,为进一步的工作打下了基础。
OBJECTIVE : To establish a fully human Fab gene library based on the chimeric HAb18 Fab antibody using the vector pComb3X and adopting the guideline selection chain replacement technique. METHODS: A full set of human Fd gene fragments and L-chain genes were amplified from peripheral blood mononuclear cells (PBMCs) from patients with hepatocellular carcinoma by RT PCR. First, the Fd gene was cloned into the display vector pComb3X/CL containing the chimeric L-chain gene, and a human-mouse hybrid Fab gene library was established. The extracellular region fragment of the prokaryotic expressed non-fusion HAb18G was used as an antigen to screen for heterozygous genes. The Fab gene was combined to obtain the human Fd gene. Then the selected Fd gene was paired with the human L-chain gene library to create a full human Fab gene library and to screen the full human Fab gene. The IPTG-induced expression of bacteria was lysed and the supernatant was used to analyze the function of the pIII Fab fusion protein and the coding gene was sequenced. RESULTS: A 2×10 7 hybrid Fab gene library was established. After 6 rounds of selection, 7 hybrid Fab genes were obtained. Using the screened human Fd gene, a full human fab gene library with a library capacity of 0.8×10 7 was established. After four rounds of screening, two human Fab genes with high affinity and strong specificity were obtained. The sequencing results showed that the two Fab genes had the same Fd gene sequence and belonged to the same IgG2 subclass as the parent antibody; the L chain gene was κ and the variable regions belonged to the Vκ3 family. Conclusion : Using guideline-selective-chain replacement technique, a specific, fully humanized anti-hepatoma antigen HAb18G Fab antibody was screened out, which laid the foundation for further work.