Construction of scFv phage display library with hapten-specific repertories and characterization of anti-ivermectin fragment isolated from the library

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• 作者：Xiao Zhang ; Cunzheng Zhang ; Yuan Liu ; Xiangyang Yu and Xianjin Liu
• 关键词：Phage display library ; scFv ; 16 ; membered macrocyclic backbone micro ; molecular substances ; Screening
• 刊名：European Food Research and Technology
• 出版年：2010
• 出版时间：July, 2010
• 年：2010
• 卷：231
• 期：3
• 页码：423-430
• 全文大小：341.7 KB

文摘

A specific phage display library was developed for screening antibodies against micro-molecular substances with 16-membered macrocyclic backbone. Through mRNA extraction, RT-PCR and gene splicing by overlap extension PCR (SOE-PCR), single-chain fragment variable (scFv) gene fragments about 750 bp were generated and ligated with the phagemid pCANTAB5E and were transformed into competent cells. A phage display library containing 2.4 ¡Á 106 clones was constructed from milbemycin oxime-bovine serum albumin (MILO-BSA) immunized mice. The screening was carried out by ivermectin-bovine serum albumin (IVM-BSA) with different concentration levels. Furthermore, scFv phage clones were isolated within the four round library panning and screening by phage- ELISA, 10 positive clones were obtained finally. These positive clones were then sequenced, and their soluble type antibodies were identified and showed significant binding activity.