从组合噬菌体展示文库中产生兔单克隆抗体片段及其在毕赤酵母中的表达

Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris.

作者信息

Ridder R, Schmitz R, Legay F, Gram H

机构信息

Sandoz Pharma, Department of Drug Metabolism and Pharmacokinetics, Basel, Switzerland.

出版信息

Biotechnology (N Y). 1995 Mar;13(3):255-60. doi: 10.1038/nbt0395-255.

DOI:10.1038/nbt0395-255

PMID:9634767

Abstract

We have applied the combinatorial immunoglobulin library and phage display technologies to generate monoclonal rabbit single-chain Fv (scFv) antibody fragments specific for recombinant human leukemia inhibitory factor (rhLIF). The B cell immunoglobulin repertoire of an immunized rabbit was immortalized by the combinatorial cloning of the rearranged variable domains of light (VL) and heavy (VH) chains. Affinity selection of the library displaying the rabbit antibody domains on the phage surface resulted in the isolation of phage encoding scFv antibodies which specifically bind to the antigen. We utilized the methylotrophic yeast Pichia pastoris for high level secretion of soluble and functional scFv antibody fragment. More than 100 mg/L of pure and functional rabbit anti-rhLIF scFv antibody was obtained directly from the P. pastoris culture supernatant by one-step affinity chromatography.

摘要

我们应用组合免疫球蛋白文库和噬菌体展示技术，生成了对重组人白血病抑制因子（rhLIF）具有特异性的单克隆兔单链Fv（scFv）抗体片段。通过对轻链（VL）和重链（VH）重排可变区的组合克隆，使免疫兔的B细胞免疫球蛋白库永生化。在噬菌体表面展示兔抗体结构域的文库进行亲和选择，从而分离出编码与抗原特异性结合的scFv抗体的噬菌体。我们利用甲基营养型酵母毕赤酵母实现可溶性和功能性scFv抗体片段的高水平分泌。通过一步亲和层析，直接从毕赤酵母培养上清中获得了超过100 mg/L的纯功能性兔抗rhLIF scFv抗体。