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Development and identification of fully human scFv-Fcs against Staphylococcus aureus

BACKGROUND: Staphylococcus aureus, a gram-positive pathogen, causes many human infections. Methicillin-resistant S. aureus (MRSA) is the most common drug-resistance bacteria. Nearly all MRSA bacteria are resistant to several drugs. Specific antibodies are the main components of the host’s humoral im...

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Autores principales: Nian, Siji, Wu, Tong, Ye, Yingchun, Wang, Xu, Xu, Wenfeng, Yuan, Qing
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850644/
https://www.ncbi.nlm.nih.gov/pubmed/27129873
http://dx.doi.org/10.1186/s12865-016-0146-z
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author Nian, Siji
Wu, Tong
Ye, Yingchun
Wang, Xu
Xu, Wenfeng
Yuan, Qing
author_facet Nian, Siji
Wu, Tong
Ye, Yingchun
Wang, Xu
Xu, Wenfeng
Yuan, Qing
author_sort Nian, Siji
collection PubMed
description BACKGROUND: Staphylococcus aureus, a gram-positive pathogen, causes many human infections. Methicillin-resistant S. aureus (MRSA) is the most common drug-resistance bacteria. Nearly all MRSA bacteria are resistant to several drugs. Specific antibodies are the main components of the host’s humoral immunity, and play a significant role in the process of the host’s resistance to bacterial infection. RESULTS: A single-chain variable fragment (scFv) library was constructed using mRNA from the peripheral blood mononuclear cells of S. aureus infected volunteers. After the scFv library DNA was transformed into Escherichia coli TG1, ~1.7 × 10(7) independent clones with full-length scFv inserts. The scFv library was screened by phage display for three rounds using S. aureus as an antigen. The single clones were chosen at random and the scFvs were expressed for enzyme-linked immunosorbent assay (ELISA) assessment. Approximately 50 % of the clones were positive with good binding activity to S. aureus. To improve the stability of scFvs, scFv-fragment crystallizable regions (-Fcs) were constructed and expressed in E. coli DH5α. The expressed scFv-Fcs were purified and identified by western blot. These antibodies were further characterized and analyzed for bioactivity. The results showed that the expression level and folding of scFv-Fcs induced at 25 °C without isopropyl β-D-1-thiogalactopyranoside (IPTG) were higher than that induced at 32 °C with 1.0 mmol/L IPTG. scFv-Fcs had good bioactivity and could specifically bind with S. aureus. CONCLUSION: scFv-Fcs against S. aureus were successfully constructed and are good candidates for the development of future adjunctive therapy for severe S. aureus infections.
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spelling pubmed-48506442016-04-30 Development and identification of fully human scFv-Fcs against Staphylococcus aureus Nian, Siji Wu, Tong Ye, Yingchun Wang, Xu Xu, Wenfeng Yuan, Qing BMC Immunol Research Article BACKGROUND: Staphylococcus aureus, a gram-positive pathogen, causes many human infections. Methicillin-resistant S. aureus (MRSA) is the most common drug-resistance bacteria. Nearly all MRSA bacteria are resistant to several drugs. Specific antibodies are the main components of the host’s humoral immunity, and play a significant role in the process of the host’s resistance to bacterial infection. RESULTS: A single-chain variable fragment (scFv) library was constructed using mRNA from the peripheral blood mononuclear cells of S. aureus infected volunteers. After the scFv library DNA was transformed into Escherichia coli TG1, ~1.7 × 10(7) independent clones with full-length scFv inserts. The scFv library was screened by phage display for three rounds using S. aureus as an antigen. The single clones were chosen at random and the scFvs were expressed for enzyme-linked immunosorbent assay (ELISA) assessment. Approximately 50 % of the clones were positive with good binding activity to S. aureus. To improve the stability of scFvs, scFv-fragment crystallizable regions (-Fcs) were constructed and expressed in E. coli DH5α. The expressed scFv-Fcs were purified and identified by western blot. These antibodies were further characterized and analyzed for bioactivity. The results showed that the expression level and folding of scFv-Fcs induced at 25 °C without isopropyl β-D-1-thiogalactopyranoside (IPTG) were higher than that induced at 32 °C with 1.0 mmol/L IPTG. scFv-Fcs had good bioactivity and could specifically bind with S. aureus. CONCLUSION: scFv-Fcs against S. aureus were successfully constructed and are good candidates for the development of future adjunctive therapy for severe S. aureus infections. BioMed Central 2016-04-29 /pmc/articles/PMC4850644/ /pubmed/27129873 http://dx.doi.org/10.1186/s12865-016-0146-z Text en © Nian et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Nian, Siji
Wu, Tong
Ye, Yingchun
Wang, Xu
Xu, Wenfeng
Yuan, Qing
Development and identification of fully human scFv-Fcs against Staphylococcus aureus
title Development and identification of fully human scFv-Fcs against Staphylococcus aureus
title_full Development and identification of fully human scFv-Fcs against Staphylococcus aureus
title_fullStr Development and identification of fully human scFv-Fcs against Staphylococcus aureus
title_full_unstemmed Development and identification of fully human scFv-Fcs against Staphylococcus aureus
title_short Development and identification of fully human scFv-Fcs against Staphylococcus aureus
title_sort development and identification of fully human scfv-fcs against staphylococcus aureus
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850644/
https://www.ncbi.nlm.nih.gov/pubmed/27129873
http://dx.doi.org/10.1186/s12865-016-0146-z
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