人源Fab段噬菌体抗体库的构建及抗IL-4抗体的初步筛选  

Construction of a Human Fab Phage Antibody Library and the Initial Screening of Phage Antibody against Interleukin-4

作　　者：胡占东[1] 公倩[2] 朱铁虹[2] 畅继武[1] 

机构地区：[1]天津医科大学第二医院泌尿外科研究所,300211 [2]天津医科大学总医院

出　　处：《天津医药》2011年第6期493-495,578,共4页Tianjin Medical Journal

基　　金：天津市科委重点项目(项目编号:034608411)

摘　　要：目的:构建大容量人源Fab段噬菌体抗体库,从中筛选抗白细胞介素(IL)-4的抗体元件,为人源抗体的制备建立平台。方法:采集18例健康成人外周血,分离淋巴细胞,从中提取总RNA,用逆转录聚合酶链反应(RT-PCR)法扩增Fab段抗体基因,插入噬菌粒pComb3XSS载体内,构建噬菌体抗体元件库,用内切酶鉴定基因片段的插入。以抗原IL-4对抗体库进行富集筛选。筛选后,随机抽取菌株通过噬菌体-酶联免疫吸附法(Phage-ELISA)进行检测,并且对阳性克隆DNA的序列进行测定。结果:构建的人源Fab段噬菌体抗体库库容为2.4×108,酶切鉴定插入基因片段大小正确,从中筛选到与IL-4有结合活性的克隆。ELISA特异鉴定阳性,测序结果证实为人免疫球蛋白可变区基因片段。结论:成功构建了大容量人源化噬菌体抗体元件库,并初步获得人源性抗IL-4-Fab抗体。Objective： To construct a large-capacity human Fab phage antibody library and screen interleukin （IL-4） antibody components to build a platform for preparation of human antibody. Methods： The peripheral blood was collected from 18 healthy adults, and lymphocytes were separated. The total RNA was extracted and Fab genes were amplified by RT-PCR. In order to construct phage antibody component library, the genes were ligated into the vector phagemid pComb3XSS. The insertion of genes was identified by restriction enzymes. In the end, the antibody library was screened using IL-4 as an antigen. After panning,strains were identified by PhageELISA, and DNA segments of positive clone were sequenced. Results： The storage capacity of human Fab segment phage antibody library was 2.4×10^8. The size of inserted gene fragment was proved correct by digestion. Binding activity clone was screened with IL-4 from phage antibody library. ELISA specific identification showed positive. The sequencing results confirmed that the gene was human immunoglobulin variable region gene. Conclusion： The large-capacity human phage antibody component library was successfully constructed. The anti-IL-4 human Fab antibody was obtained, which can be used in large scale production of human IL-4 and detection of blood levels of IL-4.

关 键 词：淋巴细胞 细菌噬菌体 遗传工程 免疫球蛋白Fab片段 白细胞介素4 逆转录聚合酶链反应 

分 类 号：R392.11[医药卫生—免疫学]