伏马菌素B_1噬菌体单链抗体库的构建与鉴定  

Construction and identification of phage ScFv library against fumonisin B_1

作　　者：王莹[1] 顾舒舒[1] 何成华[1] 王玉伟[1] 任喆 施志玉[1] 赵士侠[1] 张海彬[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《南京农业大学学报》2013年第5期113-119,共7页Journal of Nanjing Agricultural University

基　　金：农业部948项目(2012-Z22);教育部高等学校博士点基金项目(20110097110015);国家自然科学基金项目(30901091);中央高校基本科研业务费专项资金(0906J0397);江苏省农业三项工程项目(SX(2010)268)

摘　　要：从伏马菌素B1(FB1)杂交瘤细胞株F3中扩增出V H和V L基因片段,再经重叠延伸PCR(SOE-PCR)拼接扩增得到单链抗体(ScFv)基因,然后克隆到pCANTAB 5E噬菌粒载体上,转化感受态大肠杆菌TG1,并经辅助噬菌体M13K07超感染,构建FB1毒素噬菌体单链抗体库,ScFv基因插入率为100%,库容约为1.5×107,噬菌体的滴度为2.1×1015PFU·mL-1。免疫亲和筛选和富集后,经ELISA法最终筛选得到5株可分泌阳性噬菌体抗体的细菌。结论:伏马菌素B1噬菌体单链抗体库构建成功。The VH and VL gene fragments were first amplified from hybridoma cell line（ F3 ）specific to fumonisin B1 （FBj）integrated as single-chain antibody（ ScFv）gene using gene splicing by overlap extension PCR （SOE-PCR）. The ScFv was cloned into a phagemid vector pCANTAB 5E, and then was transformed into Escherichia coli TG1 to establish a phage ScFv library with the helper phage M13K07. The results indicated that the insert rate of SvFv gene was 100% ,the phage antibody contained 1.5×10^7 independent clones and phage titer was 2.1 × 10^15 PFU· mL^-1. After biopanning enrichment and screening, five strains that could secrete the FB1 specific phage single-chain antibody were selected successfully. Conclusion ：the phage ScFv library against FB~ was constructed successfully.

关 键 词：伏马菌素B1 单链抗体 噬菌体抗体库 

分 类 号：S852.4[农业科学—基础兽医学]