用体外致敏的鼻咽癌患者B细胞构建噬菌体抗独特型抗体库  

Construction of phage anti-idiotypic antibody library using sensitized in vitro B-lymphocytes of nasopharyngeal cancer patients

作　　者：何小鹃[1] 李官成[1] 朱建高[1] 李跃辉[1] 

机构地区：[1]中南大学湘雅医学院肿瘤研究所,湖南长沙410078

出　　处：《细胞与分子免疫学杂志》2003年第3期266-268,271,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目 (No .30 2 70 52 1);湖南省自然科学基金资助项目(No.0 1JJY2 0 2 1 );教育部留学回国人员科研启动基金;湖南省卫生厅基金资助

摘　　要：目的 :构建噬菌体人源抗独特型抗体库。方法 :体外致敏并用EBV转化鼻咽癌患者的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MC10 6 1,构建噬菌体呈现型ScFv库。结果 :经EBV转化的 10例鼻咽癌患者的PBMC中 ,8例有鼻咽癌抗独特型抗体产生。经多次PCR ,扩增出 5种VH(γ、μ)和 7种VL(κ、λ)基因 ,经连接组成 14种ScFv基因。将ScFv基因与载体连接后 ,导入大肠杆菌MC10 6 1。经四环素抗性筛选 ,得到库容为 1.1× 10 7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 70 %。结论 :用体外致敏法结合噬菌体抗体库技术 ,制备人源抗独特型单链抗体(Ab2 βScFv)AIM: To construct phage human anti idiotypic antibody library . METHODS: Peripheral blood mononuclear cells(PBMCs) of patients with nasopharyngeal cancer(NPC) were sensitized in vitro and transformed by Epstein Barr virus(EBV). V H and V L genes were re amplified by PCR and combined to single chain fragment of variable region(ScFv) genes. ScFv genes were cloned into vector fUSE5 and transformed into MC1061 by electroporation to construct the ScFv displaying phage library. RESULTS: Detection of Sandwich ELISA showed that of 10 NPC patients,8 patients' B cells transformed by EBV could produce anti idiotypic antibodies to NPC. 5 types of V H genes and 7 types of V L genes were obtained by PCR re amplification and then connected with (Gly 4Ser) 3 linker to form 14 types of ScFv genes. ScFv genes digested with Sfi I were cloned into vector fUSE5 and transformed into MC1061 via electroporation. Phage anti idiotypic antibody library with sink size being 1.1×10 7 was obtained through tetracycline resistant secreening . The percentage of full length ScFv gene inserted into phage DNA was 70%. CONCLUSION: A strategy for preparing human single chain anti idiotypic antibody by means of phage antibody library technique in combination with EBV transformation technique is feasible.

关 键 词：鼻咽癌 噬菌体呈现技术 抗独特型抗体 

分 类 号：R739.63[医药卫生—肿瘤]