Department of Immunobiology, Faculty of Applied Biological Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, 739-8528, Japan.
Cytotechnology. 2000 Mar;32(3):191-8. doi: 10.1023/A:1008149815908.
The chicken is a useful animal for the development of the specificantibodies against the mammalian conserved proteins. We generated twotypes of recombinant chicken monoclonal antibodies (mAbs), using a phagedisplay technique from a chicken hybridoma HUC2-13 which secreted themAb to the N-terminal of the mammalian prion protein (PrP). Althoughthe mAb HUC2-13 is a useful antibody for the prion research, thehybridoma produces a low level of antibody production. In order to producea large amount of the mAb, we have constructed a single chain fragmentvariable region (scF(V)) mAb by using the variable heavy(V(H)) and light (V(L))genes which were amplified by using the two primer pairs and theflexible linker. The two phage display mAbs (HUC2p3 and HUC2p5)expressed on a M13 filamentous phage and their soluble type mAbs(HUC2s3 and HUC2s5) were reacted with the PrP peptide antigen in theELISA. In the Western blot analysis, the mAbs HUC2p3 and HUC2s3 wereas reactive to PrP(c) from mouse brains as the mAb HUC2-13 was. The nucleotide sequences of V(H) and V(L) genes from HUC2-13 and the two cloneswere identical except for only one residue. These results indicate that themethods presented here provide an effective tool for the improvement ofthe low levels of antibody production in the chicken hybridoma system.
鸡是用于开发针对哺乳动物保守蛋白的特异性抗体的有用动物。我们使用噬菌体展示技术从分泌针对哺乳动物朊病毒蛋白（PrP）N 端的单克隆抗体（mAb）的鸡杂交瘤 HUC2-13 生成了两种类型的重组鸡单克隆抗体（mAb）。尽管 mAb HUC2-13 是朊病毒研究的有用抗体，但杂交瘤产生的抗体产量较低。为了大量生产 mAb，我们使用通过两对引物和柔性接头扩增的可变重（V（H））和轻（V（L））基因构建了单链片段可变区（scF（V））mAb。两种噬菌体展示 mAb（HUC2p3 和 HUC2p5）在 M13 丝状噬菌体上表达，其可溶性 mAb（HUC2s3 和 HUC2s5）在 ELISA 中与 PrP 肽抗原反应。在 Western blot 分析中，mAb HUC2p3 和 HUC2s3 与来自小鼠大脑的 PrP（c）的反应性与 mAb HUC2-13 相同。HUC2-13 和两个克隆的 V（H）和 V（L）基因的核苷酸序列除了一个残基外完全相同。这些结果表明，这里提出的方法为提高鸡杂交瘤系统中抗体产量的低水平提供了一种有效的工具。
