摘要
目的:从大容量噬菌体抗体库中筛选人源性抗呼吸道合胞病毒F蛋白的单链抗体。方法:以RSV F蛋白为靶抗原,通过"吸附-洗涤-洗脱-扩增"过程从天然人源性噬菌体抗体库中筛选特异性抗F蛋白单链抗体。5轮筛选后,单克隆经ELISA检测,阳性克隆进行核酸序列分析,并将阳性克隆噬菌体感染E.coliHB2151,经IPTG诱导,制备抗RSV F蛋白的可溶性单链抗体,并进行Western blot及Dot blot分析。结果:经过筛选,获得了18株能与F蛋白特异性结合的阳性克隆,取OD值最高的克隆E4经测序并检索Kabat数据库分析,显示其基因与人免疫球蛋白可变区基因具有高度同源性,Western blot及Dot blot分析表明为单链抗体。结论:利用天然人源性噬菌体抗体库技术制备出高特异性的人源性抗RSV F蛋白单链抗体。
Objective： To obtain the human single chain antibody （scFv） against fusion protein of Respiratory Syncytial Virus. Methods：A naive library of phage display human scFv, genes of which were derived from peripheral blood lymphocytes （PBLs） of 10 healthy donors, was constructed and panned by F of RSV to select specific anti-RSV F scFv. After 5 rounds of panning, the activity of individual clone was examined by ELISA. The genes of positive clones were sequenced. Soluble scFv was prepared through infecting E. coli HB2151 with one of the positive phage clones. Results： After the panning, scFvs from 18 clones can bind F specifically by ELISA. DNA sequence analysis of the clone FA with the highest OD indicates that the variable region gene of the clone is highly homologous with human immunoglobulin gene family. The expression of scFv E4 has been confirmed by Western blot and Dot blot. Conclusion：The specific human anti-RSV F scFv has been prepared successfully by exploiting phage antibody library.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2009年第9期7-11,共5页
China Biotechnology
关键词
人呼吸道合胞病毒
F蛋白
噬菌体抗体库
单链抗体
Human respiratory syncytial virus Fusion glycoprotein Phage display library Single chain fragment variable
