人-鼠杂合Fab噬菌体抗体库的建立

Construction of human-mouse hybrid Fab phage antibody libraries

目的 建立人 鼠杂合Fab噬菌体抗体库 ,经过扩增和筛选 ,得到目的抗体。方法 自肝癌患者外周血单个核细胞中扩增全套人抗体Fd片段基因 ,分别克隆入含嵌合轻链的展示载体pComb3和pComb3X中 ,建立人 鼠杂合Fab噬菌体抗体库。然后利用肝癌抗原HAb18GE进行筛选 ,并对抗体库的扩增和筛选条件进行优化。结果 pComb3展示抗体库在 37℃进行扩增挽救时存在明显的目的片段重组丢失现象。pComb3X展示抗体库在不同的扩增条件下均未发现明显的重组丢失现象。经过 4轮筛选后 ,噬菌体展示抗体ELISA显示 ,鉴定克隆与原核表达GST及无关蛋白存在明显交叉反应 ,而改用诱导表达细菌裂解上清进行ELISA鉴定可以抑制交叉反应的发生。结论 通过建立杂合Fab抗体库 ,并对扩增和筛选条件进行优化 。

Objective To construct human-mouse hybrid Fab phage antibody libraries and to select target Fab genes. Methods With Fd repertoire genes of PBMC from hepatoma patients and the chemeric light chain gene of cFab-HAb18, two hybrid Fab phage antibody libraries(pComb3 and pComb3X) were constructed. The GST fusion and non-fusion HAb18GE were used as antigens to select the target antibodies with the optimized strategies of amplifying and screening. Results When pComb3 displaying library was cultivated at 37℃, the target fragments could not be cut out with the restriction endonucleases from most clones of the library, which was an indication of “recombinant-deletion”. But no “recombinant-deletion” was found in pComb3X displaying library. Because of the serious cross-reaction, the phage-display Fab ELISA could not be used for the selection of positive clones. But positive clones could be identified in the supernatant of lysate with ELISA afterbeing induced by IPTG. Conclusion Through the construction of HuMFab phage antibody libraries and the optimization of screening strategies, we get the desired HuMFab genes for subsequent research.