摘要
目的应用噬菌体展示技术构建抗人DR5(death receptor 5,DR5)单链抗体(single chain Fv,scFv)文库,从中筛选抗DR5 scFv。方法利用重组人DR5抗原免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸PCR将VH和VL基因拼接成scFv基因,以SfiⅠ位点定向插入PAK100噬菌粒载体,转化E.coli XL1-Blue,构建了库容为1×106的抗DR5单链抗体库。对抗体库进行5轮富集筛选后,phage-ELISA检测阳性克隆的抗原特异性,取1株阳性克隆进行测序分析。结果抗DR5 scFv基因序列长747 bp,编码249个氨基酸,具有和DR5结合的特异性。结论本文利用噬菌体抗体库筛选到了抗DR5 scFv,为研制临床免疫治疗的新型抗体奠定了实验基础。
The study aim to construct single chain Fv（scFv） library against human death receptor 5 by phage display technique and to obtain the anti-DR5 scFv clones selected from the library.Total RNA was extracted from splenocytes of the Balb/c mice immunized with recombinant human DR5.Complementary DNA fragments of variable heavy（VH） and variable light（VL） chains of antibodies were prepared by RT-PCR and assembled into scFv.Then the scFv fragment was inserted into the phagemid vector PAK100 through SfiⅠsites.The phagemids containing scFv cDNA were transformed into E.coli XL1-Blue bacterial cells to generate scFv antibody library with a diversity of approximately 1×106.Five rounds of enrichment and panning were performed to select specific anti-DR5 scFv from the library.The antigen binding activity was evaluated via ELISA assay.Sequencing analysis of one positive clone showed that the anti-DR5 scFv was 747 bp,and coded 249 amino acids.The results mean an anti-DR5 scFv is obtained from scFv antibody library,which set a base for seeking novel antibodies of using in immunotherapy.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2011年第11期945-948,共4页
Immunological Journal
基金
传染病预防控制国家重点实验室开放课题(2008SKLID308)
河南省重点科技攻关资助项目(0224630174)
关键词
噬菌体展示技术
死亡受体5
单链抗体
Phage display technique
Death receptor 5
Single-chain antibody
