|Per citare questo articolo|
I TUOI DATI
I TUOI ORDINI
N. prodotti: 0
Totale ordine: € 0,00
I TUOI ABBONAMENTI
I TUOI ARTICOLI
Rivista di Biologia Molecolare e Biotecnologie
Indexed/Abstracted in: EMBASE, Science Citation Index Expanded (SciSearch), Scopus
Impact Factor 0,246
Minerva Biotecnologica 2017 March;29(1):33-40
Copyright © 2016 EDIZIONI MINERVA MEDICA
Comparison between mammalian cell and bacterial cell-free system for high-throughput expression of linear PCR-amplified immunoglobulin genes
Muhamad ALI ✉
Laboratory of Microbiology and Biotechnology, Faculty of Animal Sciences, Mataram University, Mataram, Indonesia
BACKGROUND: Monoclonal antibodies have become the most crucial and fastest growing group of protein therapeutics dedicated by modern biotechnology. These molecules represent a powerful reagent not only as a weapon to fight against lethal pathogens, but also as tools for many molecular immunology investigations. Therefore, the development of a high-throughput procedure to generate the antibodies in quite an amount is highly required. Two rapid methods, mammalian cells-based expression and bacterial cell-free transcription/translation system, have been developed for the rapid generation of a functional monoclonal antibody. However, the use of both methods to express the same linear polymerase chain reaction (PCR)-amplified immunoglobulin genes (Ig-genes) for generation of a functional monoclonal antibody fragment (Fab) has not been compared yet.
METHODS: Ig-genes were amplified from single plasma cell of an immunized mouse using MAGrahd Reactor. The PCR-amplified Ig-genes were then treated with TdT for random nucleotide tailing in the 3’-end and continued with target-selective joint PCR (TS-jPCR) to produce linear Ig-expression constructs. DNA transfection of the constructs was performed using the FuGENE HD transfection reagent into 293FT cells. At three days after transfection, the culture supernatants were analyzed for the secretion of Fab fragment. In the cell-free expression, the linear Ig-expression constructs were directly used as templates for Fab generation in E. coli cell-free expression system.
RESULTS: Mammalian cell and a bacterial cell-free system could be used to express a linear PCR-amplified Ig-genes to generate functional antibody fragment. The antigen binding activity of the mammalian cell-generated Fab fragment was 20-fold higher than those of the cell free-generated Fab fragment. However, the use of the cell-free system is faster, enabling the screening of antigen binding by ELISA within two hours.
CONCLUSIONS: This report describes the first comparison of mammalian cell and bacterial cell-free system for high-throughput expression of linear PCR-amplified immunoglobulin genes. Bacterial cell-free system entirely bypasses tedious in vivo process required for antibody production, which dramatically reduced the burden of a novel antibody discovery.
KEY WORDS: Antibodies, monoclonal - Genes, immunoglobulin - Plasma cells