大容量天然鼠源核糖体展示单链抗体文库的构建与鉴定  

Construction and identification of high-capacity,naive mouse ribosome display single chain Fv library

作　　者：程海卫[1] 陈一飞[1] 丁豪杰[1] 杨怡[1] 卓洵辉[1] 郭筱璐 陈学秋[1] 杜爱芳[1] 

机构地区：[1]浙江大学动物科学学院浙江省动物预防医学重点实验室,浙江杭州310058

出　　处：《中国兽医学报》2016年第5期763-767,777,共6页Chinese Journal of Veterinary Science

基　　金：国家"863"计划资助项目(2012AA101602)

摘　　要：为了构建大容量、天然鼠源核糖体展示(ribosome display,RD)单链抗体(single-chain fragment variant,scFv)文库。通过提取SPF级小鼠脾脏总RNA,反转录为cDNA,利用引物分别扩增鼠抗体的VH、VL和Cκ基因,采用重叠延伸PCR(SOE-PCR)方法,通过Linker((Gly4Ser)3)相互拼接,构建单链抗体scFv基因文库和核糖体展示基因文库;经连接转化,对所构建基因文库进行菌落PCR鉴定和序列测定分析。所构建核糖体展示基因文库库容量约为5.6×1013,基因片段全长大小约为1 100bp,基因文库序列具有较好的完整性和多样性。本试验成功构建了一个大容量、天然鼠源核糖体展示单链抗体文库,为进一步筛选单链抗体奠定基础。To construction of high-capacity,naive mouse ribosome display single chain Fv library.Total RNA was extracted from the spleen cell of the specific pathogen free mouse followed RTPCR using random primer.The heavy chain variable region gene（VH）,light chain variable region gene（VL）and the kappa chain constant region gene（Cκ）of the immunoglobulin were amplified respectively by PCR.They were linked by a linker encoding（Gly4Ser）3splicing and overlap extension PCR（SOE-PCR）to construct single chain Fv library and ribosome display library.The positive clones were identified by PCR,sequenced after linked and transformed to competent cells.The constructed ribosome display library showed a good integrity and diversity,with a capacity of5.6×10^13 and a gene length of about 1 100 bp.This study has constructed a high-capacity,naive mouse ribosome display single chain Fv library successfully,which would play roles in screening scFv.

关 键 词：核糖体展示 天然 鼠源 基因文库 单链抗体 

分 类 号：S85[农业科学—兽医学] Q78[农业科学—畜牧兽医]