摘要
目的构建抗肝癌人源噬菌体单链抗体库。方法体外致敏并用EBV转化肝癌患者的PBMC。用PCR分别扩增VH和VL基因并组成ScFv基因。将ScFv基因与载体连接后,转化大肠杆菌MC1061,构建噬菌体呈现型ScFv库。结果经EBV转化的4例肝癌患者PBMC,ELISA检测均有抗肝癌抗体产生,经多次PCR,扩增出6种VH(γ、μ)和9种VL(κ、λ)基因,经连接组成54种ScFv基因。将ScFv基因与载体连接后,导入大肠杆菌MC1061。经四环素抗性筛选,得到库容为1.0×108的初级噬菌体抗独特型抗体库,噬菌体DNA中全长ScFv基因的插入率为80%。结论用体外致敏法结合噬菌体抗体库技术,制备全人源抗肿瘤单链抗体(ScFv),这一策略是完全可行的。
[Objective] To construct fully humanized phage antibody hbrary. [Methods] Peripheral blood mononuclear cells （PBMCs） of patients with liver cancer were sensitized in vitro and transformed by Epstein-Barr virus （EBV）. VH and VL genes were reamplified by PCR and combined to single-chain fragment of variable region （ScFv） genes. ScFv genes were cloned into vector FUSE5 and transformed into MC1 061 by electroporation to construct the ScFv-displaying phage library. [Results] Detection of ELISA showed that 2 liver cancer patients＂ B cells transformed by EBV could produce specific antibodies to hepatoma carcinoma cell. 6 types of VH genes and 9 types of VL genes were obtained by PCR reamplification then connected with （Gly4Ser）3 linker to form 54 types of ScFv genes. ScFv genes digested with Sfi I were cloned into vector FUSE5 and transformed into MC1 061 via electroporation. Phage antibody library with sink size being 1.0×10s was obtained through tetracycline-resistant secreening. The percentage of full-length ScFv gene inserted into phage DNA was 80%. [Conclusion] A strategy for preparing fully humanized single chain antibody by means of phage antibody library technique in combination with EBV transformation technique is fully feasible.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2008年第8期1040-1044,共5页
China Journal of Modern Medicine
基金
湖南省卫生厅科研基金(A2003001)
关键词
EB病毒转化
噬菌体抗体库
肝癌
单链抗体
EBV immortalization
phage antibody libraries
liver cancer
single-chain Fv
