用EBV转化的肝癌患者B细胞构建噬菌体单链抗体库  

Construction of phage antibody library using sensitized in vitro B-lymphocytes of liver cancer patients

作　　者：肖艳[1] 李官成[1] 李跃辉[1] 童永清[1] 张志杰[1] 

机构地区：[1]中南大学肿瘤研究所免疫室,湖南长沙410078

出　　处：《中国现代医学杂志》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)

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.

关 键 词：EB病毒转化 噬菌体抗体库 肝癌 单链抗体 

分 类 号：R735.7[医药卫生—肿瘤]