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 nave 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.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
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)
