Construction of a full-length antibody phage display vector
| Autor: | Baowei Li, Johnny X. Huang, Haimei Li, Jinhua Dong, Yang Cong, Chen Limei, Liqian Zhang |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
XhoI Phage display CD3 Complex medicine.drug_class viruses Immunology Genetic Vectors Programmed Cell Death 1 Receptor Biopanning Immunoglobulin light chain Monoclonal antibody 03 medical and health sciences 0302 clinical medicine medicine Escherichia coli Immunology and Allergy Humans Mass Screening Cloning Molecular Hybridomas biology Chemistry Tumor Necrosis Factor-alpha Adalimumab Trastuzumab Molecular biology Restriction enzyme Open reading frame 030104 developmental biology Nivolumab Immunoglobulin G biology.protein Antibody Cell Surface Display Techniques 030215 immunology Bacteriophage M13 |
| Zdroj: | Journal of immunological methods. 494 |
| ISSN: | 1872-7905 |
| Popis: | Antibody phage display technology plays an important role in the development of monoclonal antibodies, humanization, and affinity evolution of antibodies. Thus far, antibody phage display mainly focuses on the display of antibody variable region or antigen-binding fragments. In this study, we constructed a new phage display system that can display full-length IgG antibodies on M13 phage. The phage display vector contains open reading frames (ORFs) encoding full-length the heavy and light chains of the antibody. NcoI/XhoI restriction enzyme sites were used to clone the variable region of the heavy chain into the heavy chain ORF, and SalI/NotI sites were used to clone the light chain variable region. SnaBI and SbfI restriction enzyme sites were designed between the cloning sites of heavy and light chains, respectively, to increase the cloning efficiency. The full-length antibodies of nivolumab against programmed death factor 1, trastuzumab against human epidermal growth factor 2, diL2K against the cluster of differentiation 3 epsilon, and adalimumab against tumor necrosis factor- alpha were displayed on phage with the vector. Phage-displayed antibodies showed their original antigen-binding activity. An amber codon shifted the vector to express IgG in non-suppressed Escherichia coli. The heavy and light chains of the E. coli-expressed antibodies could be detected through western blotting, and the antigen-binding activity was confirmed using an enzyme-linked immunosorbent assay. Biopanning was carried out with a model phage display antibody library, and the results showed that the novel phage system could be used for antibody library construction and highly efficient antibody screening. The reported system is the first full-length antibody phage display system. |
| Databáze: | OpenAIRE |
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