抗整合素β_3胞外区噬菌体抗体库的构建及筛选  

Construction and screening of phage display antibody library directed against the extracellular domain of human integrin β_3

作　　者：杨俊涛[1] 李正强[2] 王清明[1] 陈吉中[1] 范国才[1] 陈惠鹏[3] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850 [2]吉林大学分子酶学教育部重点实验室,长春130023 [3]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2004年第6期552-556,共5页Letters in Biotechnology

摘　　要：用RT-PCR的方法从人胶质瘤BT-325细胞中扩增人整合素β3胞外区,并克隆到载体pET-24a中构建表达载体。表达的人整合素β3胞外区经变性、复性和纯化后免疫BALB/c小鼠,提取脾脏总RNA,用RT-PCR技术扩增小鼠抗体重链(VH)和轻链(VL)可变区基因,经重叠PCR(SOE-PCR)将VH和VL连接成单链抗体(scFv)基因,并克隆到噬菌粒载体pCANTAB5E中,电转化至大肠杆菌TG1,经辅助噬菌体M13KO7超感染,构建噬菌体单链抗体库。通过淘选从该抗体库中筛选特异性识别人整合素β3胞外区的噬菌体单链抗体。结果表明,成功构建了库容为2.6×106的抗人整合素β3胞外区的单链抗体库,初步筛选到了与人整合素β3胞外区特异性结合的单链抗体。To construct a phage display single-chain variable fragment(scFv) library directed against the extracellular domain of human integrin β3 and screen for specific antibodies to it from the library. The mRNA isolated from human BT-325 cells was used to amplified the gene of the extracellular domain of human integrin β3 by RT-PCR. The expression vector of the extracellular domain of human integrin β3 was constructed and used to transform E.coli BL21. The extracellular domain of human integrin β3 was expressed, refolded and purified, and then was used to immunize BALB/c mice. Total splenic RNA of the immunized mice was isolated and used to amplify the heavy-chain and light-chain variable region genes of antibodies by RT-PCR. The heavy-chain and light-chain variable region genes of antibodies were then joined into a single chain by overlapping PCR with a linker DNA encoding the peptide(Gly4Ser)3. The assembled scFv fragments were cloned into the phagemids(pCANTAB5E), and the recombinant phagemids were used to transform the competent E.coli TG1. The transformed TG1 were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The specific antibodies against the extracellular domain of human integrin β3 were enriched by four rounds of panning. The positive recombinant phages were analyzed by ELISA. The extracellular domain of human integrin β3 was overexpressed. An antibody library containing 2.6×106 different clones was obtained. The phage isolates with the binding abilities and specificity for extracellular domain of human integrin β3 were selected.

关 键 词：整合素Β3 单链抗体 胞外区 噬菌体抗体库 扩增 胶质瘤 ET 电转化 重叠PCR 特异性识别 

分 类 号：Q78[生物学—分子生物学] R392[医药卫生—免疫学]