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.
出处
《军事医学科学院院刊》
CSCD
北大核心
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)
