一种大容量天然人源核糖体展示单链抗体文库的构建  

Construction of a large naive human single chain Fv (scFv) library for ribosome display

作　　者：陈锐[1] 刘友生[1] 朱江[1] 段光杰[1] 葛晓冬[1] 

机构地区：[1]第三军医大学西南医院病理科,重庆400038

出　　处：《重庆医学》2009年第9期1053-1055,共3页Chongqing medicine

基　　金：国家自然科学基金资助项目(30571924)

摘　　要：目的构建大容量天然人源核糖体展示单链抗体(single chain Fv,scFv)文库,为抗体研究提供方便有效的技术平台。方法收集健康献血志愿者的白细胞悬液,离心分离单个核细胞,抽提其总RNA,逆转录成cDNA、PCR及重叠延伸PCR法构建scFv文库,并通过第2次重叠延伸PCR在scFv DNA序列5′端添加T7启动子、茎环结构、核糖体结合位点,其3′端添加6×His标签、间隔序列(基因Ⅲ)、茎环结构等改造成核糖体展示天然人源scFv文库。结果成功构建了可用于筛选抗体的核糖体展示文库容量达1013以上。结论成功构建了大容量天然人源核糖体展示scFv文库,为筛选到高亲和力和特异性抗体提供了技术平台。Objective To construct a large naive human single chain Fv（scFv） library for ribosome display,which provides a convenient and effective platform for antibody research. Methods White blood cells suspension of a healthy donor were collected and centrifuged to isolate mononuelear cells. And total RNA of mononuclear cells were isolated and reverse transcribed to construct scFv library by PCR and overlap extension PCR amplification. A second overlap extension PCR amplification was used to add T7 promoter, stem loop structure,ribosome binding sites to the 5′ end and 6 × His tag, spacer（gene Ⅲ ）, stem loop structure to the 3′ end of scFv DNA sequence. In this way a naive scFv library for ribosome display was constructed. Results A large naive human scFv library containing ）10^13 members for ribosome display was successfully constructed. Conclusion The large naive human seFv library for ribosome display will be a useful platform for selection of antibodies with high affinity and specificity.

关 键 词：核糖体展示 文库 天然人源单链Fv(scFv)抗体 

分 类 号：Q785[生物学—分子生物学] R392.11[医药卫生—免疫学]