Cargando…
A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage
Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in rese...
Autores principales: | , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier B.V.
2007
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112517/ https://www.ncbi.nlm.nih.gov/pubmed/17360045 http://dx.doi.org/10.1016/j.vetimm.2007.02.001 |
_version_ | 1783513489956929536 |
---|---|
author | Lee, Yu-Ching Leu, Sy-Jye C. Hung, Han-Chang Wu, Hsueh-Hsia Huang, I.-Jen Hsieh, Wen-Shyang Chiu, Wen-Ta Hsieh, Ming-Song Cheng, Tsui-Fen Yang, Yi-Yuan |
author_facet | Lee, Yu-Ching Leu, Sy-Jye C. Hung, Han-Chang Wu, Hsueh-Hsia Huang, I.-Jen Hsieh, Wen-Shyang Chiu, Wen-Ta Hsieh, Ming-Song Cheng, Tsui-Fen Yang, Yi-Yuan |
author_sort | Lee, Yu-Ching |
collection | PubMed |
description | Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. In this study, phage-expressing chicken monoclonal scFv antibody was chosen and characterized with phage display antibody technology. Truncated fragments of SARS-CoV spike protein were cloned in pET-21 vector and expressed in BL-21 Escherichia coli (E. coli) cells. After purification, the purity of these recombinant spike proteins was examined on SDS–PAGE and their identity verified with Western blot analysis using anti-his antibodies and sera from convalescent stage SARS-CoV-infected patients. Using these bacteria-derived proteins to immunize chickens, it was found that polyclonal IgY antibodies in the egg yolk and sera were highly reactive to the immunogens, as shown by Western blot and immunocytochemical staining analysis. A phage displaying scFv library was also established from spleen B cells of immunized chicken with 5 × 10(7) clones. After four panning cycles, the eluted phage titer showed a 10-fold increase. In sequence analysis with chicken germline gene, five phage clones reacted, with large dissimilarities of between 31 and 62%, in the complementarity-determining regions, one dominant phage 4S1 had strong binding to fragment Se-e, located between amino acid residues 456–650 of the spike protein and this particular phage had significantly strong binding to SARS-CoV-infected Vero E6 cells. Based on the results, we conclude that generating specific scFv-expressing phage binders with the phage display system can be successfully achieved and that this knowledge can be applied in clinical or academic research. |
format | Online Article Text |
id | pubmed-7112517 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | Elsevier B.V. |
record_format | MEDLINE/PubMed |
spelling | pubmed-71125172020-04-02 A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage Lee, Yu-Ching Leu, Sy-Jye C. Hung, Han-Chang Wu, Hsueh-Hsia Huang, I.-Jen Hsieh, Wen-Shyang Chiu, Wen-Ta Hsieh, Ming-Song Cheng, Tsui-Fen Yang, Yi-Yuan Vet Immunol Immunopathol Article Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. In this study, phage-expressing chicken monoclonal scFv antibody was chosen and characterized with phage display antibody technology. Truncated fragments of SARS-CoV spike protein were cloned in pET-21 vector and expressed in BL-21 Escherichia coli (E. coli) cells. After purification, the purity of these recombinant spike proteins was examined on SDS–PAGE and their identity verified with Western blot analysis using anti-his antibodies and sera from convalescent stage SARS-CoV-infected patients. Using these bacteria-derived proteins to immunize chickens, it was found that polyclonal IgY antibodies in the egg yolk and sera were highly reactive to the immunogens, as shown by Western blot and immunocytochemical staining analysis. A phage displaying scFv library was also established from spleen B cells of immunized chicken with 5 × 10(7) clones. After four panning cycles, the eluted phage titer showed a 10-fold increase. In sequence analysis with chicken germline gene, five phage clones reacted, with large dissimilarities of between 31 and 62%, in the complementarity-determining regions, one dominant phage 4S1 had strong binding to fragment Se-e, located between amino acid residues 456–650 of the spike protein and this particular phage had significantly strong binding to SARS-CoV-infected Vero E6 cells. Based on the results, we conclude that generating specific scFv-expressing phage binders with the phage display system can be successfully achieved and that this knowledge can be applied in clinical or academic research. Elsevier B.V. 2007-05-15 2007-02-12 /pmc/articles/PMC7112517/ /pubmed/17360045 http://dx.doi.org/10.1016/j.vetimm.2007.02.001 Text en Copyright © 2007 Elsevier B.V. All rights reserved. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. |
spellingShingle | Article Lee, Yu-Ching Leu, Sy-Jye C. Hung, Han-Chang Wu, Hsueh-Hsia Huang, I.-Jen Hsieh, Wen-Shyang Chiu, Wen-Ta Hsieh, Ming-Song Cheng, Tsui-Fen Yang, Yi-Yuan A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage |
title | A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage |
title_full | A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage |
title_fullStr | A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage |
title_full_unstemmed | A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage |
title_short | A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage |
title_sort | dominant antigenic epitope on sars-cov spike protein identified by an avian single-chain variable fragment (scfv)-expressing phage |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112517/ https://www.ncbi.nlm.nih.gov/pubmed/17360045 http://dx.doi.org/10.1016/j.vetimm.2007.02.001 |
work_keys_str_mv | AT leeyuching adominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT leusyjyec adominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT hunghanchang adominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT wuhsuehhsia adominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT huangijen adominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT hsiehwenshyang adominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT chiuwenta adominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT hsiehmingsong adominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT chengtsuifen adominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT yangyiyuan adominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT leeyuching dominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT leusyjyec dominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT hunghanchang dominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT wuhsuehhsia dominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT huangijen dominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT hsiehwenshyang dominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT chiuwenta dominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT hsiehmingsong dominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT chengtsuifen dominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage AT yangyiyuan dominantantigenicepitopeonsarscovspikeproteinidentifiedbyanaviansinglechainvariablefragmentscfvexpressingphage |