人源化Fab段噬菌体抗体库的构建及初步鉴定  

Construction and primary identification of a naive Fab fragment phage library

作　　者：公倩[1] 李常颖[1] 畅继武[2] 朱铁虹[1] 

机构地区：[1]天津医科大学总医院内分泌科,天津300052 [2]天津医科大学第二医院泌尿外科研究所,天津300211

出　　处：《中国免疫学杂志》2010年第7期646-650,共5页Chinese Journal of Immunology

基　　金：天津市自然科学基金资助项目(05YFMGCO4000)

摘　　要：目的:构建人天然Fab段噬菌体抗体库,为人源抗体的制备建立技术平台。方法:从正常健康人的外周血淋巴细胞中提取总RNA,用RT-PCR方法扩增人抗体轻链和重链Fd段基因,构建噬菌体抗体库,酶切鉴定有无插入片段并测序分析,用IL-2和地高辛进行富集筛选。结果:最终构建的抗体库库容约为8.4×107,酶切鉴定显示有插入片段,抗体库重组率是70%,DNA测序分析证实抗体重链属IgG亚类,轻链为λ链。用IL-2和地高辛进行三轮筛选,抗体库均得到了不同程度的富集。结论:成功构建了一个天然的人源化噬菌体抗体库,可用于下一步胰淀素人源化抗体的筛选、纯化和表达。Objective：To construct a human Fab fragment phage display library and provide a platform for human antibody preparation.Methods：Peripheral blood lymphocytes were collected from healthy donor.The heavy chain Fd fragment and light chain of human immunoglobulin＇s genes were amplified by RT-PCR,and then cloned into phagemid pComb3XSS to generate human phage antibody library.Cutting with endonucleases such as SacⅠ,XbaⅠ,XhoⅠand SpeⅠto identify the insertion of the light chain or heavy chain Fd genes.IL-2 and digoxin as the antigen was used to scan the phage antibody library.Results：A phage antibody library of Fab had 8.4×107 members and it＇s recombinant rate was 70%.Through DNA sequencing of one positive clone,it was showed that its heavy chain belonged to IgG subvariety and its light chain to λ family.Conclusion：The success of constructing a nave human phage antibody library proves the useful of phage display system in human antibody preparation,and it can be used to select,purify and express of amylin Fab antibody.

关 键 词：噬菌体抗体库 抗体FAB段 人源化抗体 

分 类 号：R446.62[医药卫生—诊断学]