Yamanaka H I, Kirii Y, Ohmoto H
Kanebo Institute for Cancer Research, Osaka.
J Biochem. 1995 Jun;117(6):1218-27. doi: 10.1093/oxfordjournals.jbchem.a124847.
An improved combinatorial library system to raise mouse monoclonal antibodies was constructed. PCR primers have been newly designed to optimize the reaction for Pfu DNA polymerase, which has proofreading activity. The phagemid vector (pPDS) is designed to accommodate VH and Vk cDNAs, which had previously been assembled by PCR either in single chain fragment of variable regions (scFv) or Fab form. Antibody cloned in scFv form can be converted to Fab form by substituting the scFv linker of (Gly4Ser)3 with a fragment containing murine CH1 cDNA. This vector will produce soluble Fab in non-amber suppressor cells and allow the shuffling of light chains against a heavy chain. Hybridoma cell lines producing anti-human procollagenase monoclonal antibodies were used as the source of antibody mRNA. Antigen-binding ability of both scFv- and Fab-displaying phage was confirmed by ELISA against human procollagenase. They were also analyzed by DNA sequencing to verify the fidelity of Pfu DNA polymerase and to identify the primer incorporated. The mutation rate was considerably reduced compared to the mutation rate achieved by Taq DNA polymerase. Primers are incorporated into target sequences in most cases.
构建了一种改进的用于产生小鼠单克隆抗体的组合文库系统。新设计了PCR引物以优化具有校对活性的Pfu DNA聚合酶的反应。噬菌粒载体（pPDS）设计用于容纳VH和Vk cDNA，它们先前已通过PCR组装成可变区单链片段（scFv）或Fab形式。以scFv形式克隆的抗体可以通过用包含鼠CH1 cDNA的片段替代（Gly4Ser）3的scFv接头而转化为Fab形式。该载体将在非琥珀抑制细胞中产生可溶性Fab，并允许轻链与重链进行改组。产生抗人原胶原酶单克隆抗体的杂交瘤细胞系用作抗体mRNA的来源。通过针对人原胶原酶的ELISA确认了展示scFv和Fab的噬菌体的抗原结合能力。还通过DNA测序对它们进行分析，以验证Pfu DNA聚合酶的保真度并鉴定掺入的引物。与Taq DNA聚合酶实现的突变率相比，突变率大大降低。在大多数情况下，引物会掺入目标序列中。
