利用噬菌体表面展示技术克隆抗人DR5抗体可变区基因  

Clone of the anti-DR5 antibody variable sequence by phage surface display

作　　者：刘瑞敏[1] 王明丽[1] 季泽俊[1] 刘峰涛[1] 白慧玲[1] 马远方[1] 

机构地区：[1]河南大学细胞与免疫学重点实验室,河南开封475004

出　　处：《河南大学学报（医学版）》2011年第1期51-53,共3页Journal of Henan University:Medical Science

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)

摘　　要：目的:利用噬菌体表面展示技术制备抗人DR5单链抗体。方法:从抗DR5杂交瘤细胞中提取总RNA,RT-PCR扩增VH和VL基因,并利用重叠扩增PCR将VH和VL拼接为scFv基因。将scFv与PAK100载体通过相同的酶切位点连接后,转化大肠杆菌XL1-Blue,经VSCM13辅助噬菌体拯救,构建成鼠抗人DR5单链抗体库。以DR5作为固相筛选分子,对噬菌体展示肽库进行3轮"吸附-洗脱-扩增"生物筛选,从第3轮洗脱物中随机挑选10个单克隆噬菌体扩增后进行ELISA鉴定,对所筛选克隆进行DNA序列测定和通过phage-ELISA验证噬菌体单链抗体的结合特异性。结果:经过3轮筛选后,对随机选取的10个噬菌体克隆进行鉴定,其中4个具有和DR5结合的特异性。结论:利用噬菌体表面展示技术筛选单链抗体,结果更加可信。Objective：To obtain the gene sequence of antibody against human death receptor 5 by the phage surface display.Methods：By the technique of phage display,the heavy chain and light chain variable region genes（VH and VL） were assembled into single chain antibodies（scFv） with overlap extension reaction by L inker primer mix.The scFv were cloned into PAK100 phagemid and introduced into E.coli XL1-Blue.The phagemid containing bacterial colonies were infected with VSCM13,then the total surface display library of anti-human-death receptor 5 was established.The library was screened by death receptor 5 to get the antigen-specific phage clones.Positive clones were characterized by DNA sequencing.Results：After three panning rounds of＂binding-elution-amplification＂,10 individual clones were randomly selected and identified by enzyme-immunosorbent assay（ELISA）,4 of which has the characteristic of DR5.Conclusion：The technique of phage display is a more creditable method to obtain antibody gene sequence.

关 键 词：噬菌体展示技术 单链抗体 死亡受体5 

分 类 号：R392.11[医药卫生—免疫学]