用体外致敏的鼻咽癌患者B细胞构建全人源噬菌体单链抗体库  

Construction of human phage ScFv library using in vitro sensitized B-lymphocytes from nasopharyngeal cancer patients

作　　者：赵艳[1] 李跃辉[1] 黄建[1] 李官成[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：《中国医师杂志》2008年第1期30-33,共4页Journal of Chinese Physician

摘　　要：目的构建全人源抗鼻咽癌噬菌体单链抗体库，为筛选鼻咽癌相关抗原的抗体奠定基础。方法采用体外致敏与EB病毒（EBV）转化鼻咽癌患者的外周血单个核细胞（PBMC），提取总RNA，用RT—PCR技术扩增人抗体重链可变区VH基因和轻链可变区VL基因，用编码（Gly4Ser）3的互补序列连接成单链抗体ScFv基因（VH-linker—VL），并克隆到噬菌粒载体FUSE5，转化大肠杆菌MC1061，构建噬菌体呈现型ScFv库。结果成功地构建了全人源抗鼻咽癌噬菌体单链抗体库，库容量为6．5×10^7，约100％的噬菌体基因中有ScFv基因的插入。结论联合应用体外致敏和EBV转化及噬菌体呈现技术构建全人源抗鼻咽癌噬菌体单链抗体库是可行的，这为进一步筛选鼻咽癌相关抗原的抗体奠定了基础。Objective To obtain the nasopharyngeal carcinoma （NPC） cells-specific antibody, a phage display single chain antibody library was constructed. Methods Peripheral blood mononuclear cells （PBMCs） of patients with NPC were immunized in vitro by NPC cells and transformed by Epstein-Barr virus （EBV）. The total RNA was isolated and used to amplify Vn and VL genes by RT-PCR. VH and VL genes were joined with a DNA linker encoding peptide （Gly4 Ser）3 as a single chain variable fragments （ScFv）. Then, ScFv fragments were cloned into phagemids （FUSES） and the recombinant phagemids were used to transform Elcoli MC1061 to construct the svFv-displaying phage library. Results A phage display antibody library containing 6. 5 × 10^7 different clones was obtained, with 100 % of the phagemids containing ScFv gene insertion as demonstrated by PCR. Conclusions It is a feasible strategy for preparing ScFv phage display library against NPC cells by immunization in vitro, EBV transformation and phage display technique, which provide a way for screening specific antibody against NPC.

关 键 词：鼻咽肿瘤 B淋巴细胞 细菌噬菌体 肽库 

分 类 号：R739.63[医药卫生—肿瘤]