大容量天然噬菌体抗体库的建立及多样性初步验证  

Construction and preliminary identification of a large Nayve human antibody library

作　　者：王辉[1] 黄英[1] 王琰[2] 沈倍奋[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]海军总医院,北京100037

出　　处：《军事医学科学院院刊》2008年第4期305-308,358,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：“十一五”“863”重大项目(2006AA02A254)

摘　　要：目的:构建超大容量天然噬菌体抗体库。方法:从正常人外周血和新生儿脐血中分离淋巴细胞(>180份),提取RNA,用RT-PCR分别扩增抗体可变区轻重链基因(VH和VL),通过重叠PCR技术将VH和VL连接为单链抗体ScFv形式,克隆插入到pDF噬菌粒载体,转化XL1-Blue细菌得到ScFv初级抗体库,并以高感染复数(MOI≥100)感染Cre+菌株BS1365,利用Cre/LoxP位点特异性重组原理,使VH和VL基因定向同源重组匹配,随后以低感染复数(MOI<1)感染XL1-Blue,获取次级工作库。分别用5种不同抗原进行筛选,所获阳性克隆送测序以获取抗体基因。结果:抗体V区基因得到有效扩增,初级库库容3.6×107,工作库容1.8×1011,5种不同抗原筛选均得到特异性结合噬菌体抗体;测序结果表明,所获取抗体涵盖了不同的基因亚群,进一步证明抗体库具有良好的多样性。结论:经Cre/Loxp定位重组系统成功构建了超大容量天然噬菌体抗体库,初步尝试对5种抗原进行筛选均获成功,提示该抗体库多样性较好,可用于制备人源抗体。Objective：To construct a large Naive human antibody library. Methods： Diverse VL and VH genes were amplified by RT-PCR from lymphocytes collected from adult peripheral blood and newborn cord blood （ 〉 180）. The V genes were spliced to form ScFv by overlap PCR and cloned into vector pDF, obtaining a primary library. By infecting the phagemid into Cre^＋ bacteria BS1365 at high multiplicity of infection （MOI≥ 100）, the VH genes were shuffled to create a very large phage antibody library. This library was validated by selecting human antibodies against five different protein antigens. Results： The library was calculated as having a diversity of 1.8 × 10^11. Diverse specific human ScFvs against all the 5 tested antigens were obtained from the library respectively by biopanning. Conclusion：Based on Cre/Loxp system,a very large phage antibody library was constructed successfully. The success of biopanning suggests that the constructed phage antibody library can be used in human antibody preparation.

关 键 词：噬菌体抗体库 CRE/LOXP重组系统 单链抗体 

分 类 号：R392[医药卫生—免疫学]