Cargando…

Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems

BACKGROUND: Recombinant Modified Vaccinia Virus Ankara has been employed as a safe and potent viral vector vaccine against infectious diseases and cancer. We generated recMVAs encoding norovirus GII.4 genotype capsid protein by using a marker-based approach and a BAC-based system. In the marker-base...

Descripción completa

Detalles Bibliográficos
Autores principales: Kugler, Franziska, Drexler, Ingo, Protzer, Ulrike, Hoffmann, Dieter, Moeini, Hassan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688233/
https://www.ncbi.nlm.nih.gov/pubmed/31399106
http://dx.doi.org/10.1186/s12985-019-1212-y
_version_ 1783442843776319488
author Kugler, Franziska
Drexler, Ingo
Protzer, Ulrike
Hoffmann, Dieter
Moeini, Hassan
author_facet Kugler, Franziska
Drexler, Ingo
Protzer, Ulrike
Hoffmann, Dieter
Moeini, Hassan
author_sort Kugler, Franziska
collection PubMed
description BACKGROUND: Recombinant Modified Vaccinia Virus Ankara has been employed as a safe and potent viral vector vaccine against infectious diseases and cancer. We generated recMVAs encoding norovirus GII.4 genotype capsid protein by using a marker-based approach and a BAC-based system. In the marker-based approach, the capsid gene together with a reporter gene was introduced into the MVA genome in DF-1 cells. Several rounds of plaque purification were carried out to get rid of the WT-MVA. In the BAC-based approach, recMVA-BAC was produced by en passant recombineering in E. coli. Subsequently, the recMVAs were rescued in DF-1 cells using a helper rabbit fibroma virus. The BAC backbone and the helper virus were eliminated by passaging in DF-1 cells. Biochemical characteristics of the recMVAs were studied. RESULTS: We found the purification of the rare spontaneous recombinants time-consuming in the marker-based system. In contrast, the BAC-based system rapidly inserted the gene of interest in E. coli by en passant recombineering before virion production in DF-1 cells. The elimination of the reporter gene was found to be faster and more efficient in the BAC-based approach. With Western blotting and electron microscopy, we could prove successful capsid protein expression and proper virus-assembly, respectively. The MVA-BAC produced higher recombinant virus titers and infected DF-1 cells more efficiently. CONCLUSIONS: Comparing both methods, we conclude that, in contrast to the tedious and time-consuming traditional method, the MVA-BAC system allows us to quickly generate high titer recMVAs.
format Online
Article
Text
id pubmed-6688233
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-66882332019-08-14 Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems Kugler, Franziska Drexler, Ingo Protzer, Ulrike Hoffmann, Dieter Moeini, Hassan Virol J Methodology BACKGROUND: Recombinant Modified Vaccinia Virus Ankara has been employed as a safe and potent viral vector vaccine against infectious diseases and cancer. We generated recMVAs encoding norovirus GII.4 genotype capsid protein by using a marker-based approach and a BAC-based system. In the marker-based approach, the capsid gene together with a reporter gene was introduced into the MVA genome in DF-1 cells. Several rounds of plaque purification were carried out to get rid of the WT-MVA. In the BAC-based approach, recMVA-BAC was produced by en passant recombineering in E. coli. Subsequently, the recMVAs were rescued in DF-1 cells using a helper rabbit fibroma virus. The BAC backbone and the helper virus were eliminated by passaging in DF-1 cells. Biochemical characteristics of the recMVAs were studied. RESULTS: We found the purification of the rare spontaneous recombinants time-consuming in the marker-based system. In contrast, the BAC-based system rapidly inserted the gene of interest in E. coli by en passant recombineering before virion production in DF-1 cells. The elimination of the reporter gene was found to be faster and more efficient in the BAC-based approach. With Western blotting and electron microscopy, we could prove successful capsid protein expression and proper virus-assembly, respectively. The MVA-BAC produced higher recombinant virus titers and infected DF-1 cells more efficiently. CONCLUSIONS: Comparing both methods, we conclude that, in contrast to the tedious and time-consuming traditional method, the MVA-BAC system allows us to quickly generate high titer recMVAs. BioMed Central 2019-08-09 /pmc/articles/PMC6688233/ /pubmed/31399106 http://dx.doi.org/10.1186/s12985-019-1212-y Text en © The Author(s). 2019 Open Access This 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 Methodology
Kugler, Franziska
Drexler, Ingo
Protzer, Ulrike
Hoffmann, Dieter
Moeini, Hassan
Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems
title Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems
title_full Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems
title_fullStr Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems
title_full_unstemmed Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems
title_short Generation of recombinant MVA-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems
title_sort generation of recombinant mva-norovirus: a comparison study of bacterial artificial chromosome- and marker-based systems
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688233/
https://www.ncbi.nlm.nih.gov/pubmed/31399106
http://dx.doi.org/10.1186/s12985-019-1212-y
work_keys_str_mv AT kuglerfranziska generationofrecombinantmvanorovirusacomparisonstudyofbacterialartificialchromosomeandmarkerbasedsystems
AT drexleringo generationofrecombinantmvanorovirusacomparisonstudyofbacterialartificialchromosomeandmarkerbasedsystems
AT protzerulrike generationofrecombinantmvanorovirusacomparisonstudyofbacterialartificialchromosomeandmarkerbasedsystems
AT hoffmanndieter generationofrecombinantmvanorovirusacomparisonstudyofbacterialartificialchromosomeandmarkerbasedsystems
AT moeinihassan generationofrecombinantmvanorovirusacomparisonstudyofbacterialartificialchromosomeandmarkerbasedsystems