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A Neutralization scFv Antibody against IL-1 beta Isolated from a NIPA-based Bacterial Display Library

文献类型： 外文期刊

作者： Li, Tianhe 1 ; Xu, Liming 2 ; Ren, Guiping 1 ; Yin, Chengkai 1 ; Zhou, Bing 1 ; Ye, Xianlong 1 ; Li, Qingcui 1 ; Li, Ning 3 ;

作者机构： 1.Northeast Agr Univ, Coll Life Sci, Biopharmaceut Lab, Harbin 150030, Peoples R China

2.Chinese Acad Fishery Sci, Heilongjiang River Fishery Res Inst, Harbin, Peoples R China

3.SIPO, Patent Examinat Cooperat Ctr, Patent Off, Beijing, Peoples R China

关键词： scFv combinatorial bacterial display library;Modified NIPA-based bacterial display system;FACS;IL-1 beta;Inclusion body refolding;Neutralizing activity

期刊名称：CURRENT PHARMACEUTICAL BIOTECHNOLOGY （ 影响因子：2.837； 五年影响因子：2.626 ）

ISSN： 1389-2010

年卷期： 2013 年 14 卷 6 期

页码：

收录情况： SCI

摘要： Objective: RA is one of autoimmune diseases, has drawn great attention of the world. Currently, the anti- IL-1 beta monoclonal antibody Canakinumab (ACZ885) for treatment of RA has entered into clinical trials. However, Full length antibody has large molecular weight, and is difficult to penetrate the tissue or the nidus. In contrast, scFv has low molecular weight and strong penetration ability, and is favorable to increase the drug concentration in the indus, hence improving the efficacy of the drug. The aim of this study is to obtain a neutralizing scFv antibody from a combinatorial scFv library against hIL-1 beta by the modified NLPA-based bacterial display system, for further development of the small molecule antibody drug for treatment of RA. Methods: The modified NIPA-based bacterial display system was used to construct the combinatorial scFv library derived from the spleen cDNA of immunized mice with hIL-1 beta. FACS was used to screen hIL-1 beta-binding clones with FITC-labeled hIL-1 beta protein. Three clones were randomly selected from the third round of screening, and their nucleotide sequences were aligned with mouse immunoglobulin genes. The single chain antibody genes of the hIL-1 beta-binding clones were subcloned into the prokaryotic expression vector pET-27b for expression. The molecular mass of the purified anti-hIL-1 beta single chain antibody was about 28ku. The hIL-1 beta-binding ability of antibody were examined by ELISA and Western blot assays. Ability of the scFv antibody to neutralize hIL-1 beta was evaluated by the MTT test. Conclusions: In this study, it is the first time to use the NIPA-based bacteria display system to construct and screen the combinatorial scFv library. Three scFvs against hIL-1 beta were obtained from the scFv library of the immunized mice. Prokaryotically expressed and purified scFvs demonstrate binding ability with hIL-1 beta. Among the three clones. The MTT test suggests that scFv-20 is a neutralization antibody against hIL-1 beta. The study provides a lead candidate for further development of small molecule therapeutic antibodies for treatment of RA.

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