Screening and characterization of human anti-GPC3 single chain Fv antibody fragment selected by phage display
10.16438/j.0513-4870.2017-0687
- VernacularTitle:噬菌体展示全人源抗GPC3的单链抗体的筛选及鉴定
- Author:
Jia-hao JIANG
1
;
Fu-mou SUN
1
;
Yue HAN
1
;
Yang WANG
1
;
Jia-ling CAI
1
;
Min WANG
1
;
Juan ZHANG
1
Author Information
1. College of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
- Publication Type:ORIGINAL ARTICLES
- Keywords:
phage display;
single-chain variable fragment;
glypican-3;
tumor targeting
- From:
Acta Pharmaceutica Sinica
2017;52(12):1877-1883
- CountryChina
- Language:Chinese
-
Abstract:
The aim of the experiments is to screen human single-chain variable fragment (scFv) targeting glypican 3 from phage display library, and analyze its biological activity. After several rounds of panning, the binders with high affinity were obtained through phage ELISA and IMGT analysis. The desired scFv gene was then ligated with pET-22b vector yielding recombinant plasmids, which was then introduced into E. coli Rosetta (DE3). Soluble scFv protein was expressed and further purified using Ni2+ affinity chromatography. The purified proteins were identified by SDS-PAGE and Western blot. Subsequently, the affinity and cell based binding activity were measured using Surface Plasmon Resonance (SPR) and flow cytometry assay, separately. Four enriched sequences with relatively high binding affinity were found (1F7, 1D7, 1D4 and 1B10). We also found that 1F7 scFv showed better targeting ability and higher affinity. The scFv could pave the way for new immunotherapies, such as bispecific anitbody, antibody-drug conjugate and chimeric antigen receptor T-cell immunotherapy cell, etc.
