Cargando…
Design, Construction and Cloning of Truncated ORF2 and tPA(sp)-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector
BACKGROUND: Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal. OBJECTIVES: The aim of this study was to construct tPA(sp)-PADRE-truncat...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Kowsar
2015
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744464/ https://www.ncbi.nlm.nih.gov/pubmed/26865938 http://dx.doi.org/10.5812/jjm.26035 |
_version_ | 1782414497148829696 |
---|---|
author | Farshadpour, Fatemeh Makvandi, Manoochehr Taherkhani, Reza |
author_facet | Farshadpour, Fatemeh Makvandi, Manoochehr Taherkhani, Reza |
author_sort | Farshadpour, Fatemeh |
collection | PubMed |
description | BACKGROUND: Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal. OBJECTIVES: The aim of this study was to construct tPA(sp)-PADRE-truncated open reading frame 2 (ORF2) and truncated ORF2 DNA plasmid, which can assist future studies with the preparation of an effective vaccine against Hepatitis E Virus. MATERIALS AND METHODS: A synthetic codon-optimized gene cassette encoding tPA(sp)-PADRE-truncated ORF2 protein was designed, constructed and analyzed by some bioinformatics software. Furthermore, a codon-optimized truncated ORF2 gene was amplified by the polymerase chain reaction (PCR), with a specific primer from the previous construct. The constructs were sub-cloned in the pVAX1 expression vector and finally expressed in eukaryotic cells. RESULTS: Sequence analysis and bioinformatics studies of the codon-optimized gene cassette revealed that codon adaptation index (CAI), GC content, and frequency of optimal codon usage (Fop) value were improved, and performance of the secretory signal was confirmed. Cloning and sub-cloning of the tPA(sp)-PADRE-truncated ORF2 gene cassette and truncated ORF2 gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pVAX-tPA(sp)-PADRE-truncated ORF2 ((aa 112-660)) and pVAX-truncated ORF2 ((aa 112-660)). The expression of truncated ORF2 protein in eukaryotic cells was approved by an Immunofluorescence assay (IFA) and the reverse transcriptase polymerase chain reaction (RT-PCR) method. CONCLUSIONS: The results of this study demonstrated that the tPA(sp)-PADRE-truncated ORF2 gene cassette and the truncated ORF2 gene in recombinant plasmids are successfully expressed in eukaryotic cells. The immunogenicity of the two recombinant plasmids with different formulations will be evaluated as a novel DNA vaccine in future investigations. |
format | Online Article Text |
id | pubmed-4744464 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Kowsar |
record_format | MEDLINE/PubMed |
spelling | pubmed-47444642016-02-10 Design, Construction and Cloning of Truncated ORF2 and tPA(sp)-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector Farshadpour, Fatemeh Makvandi, Manoochehr Taherkhani, Reza Jundishapur J Microbiol Research Article BACKGROUND: Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal. OBJECTIVES: The aim of this study was to construct tPA(sp)-PADRE-truncated open reading frame 2 (ORF2) and truncated ORF2 DNA plasmid, which can assist future studies with the preparation of an effective vaccine against Hepatitis E Virus. MATERIALS AND METHODS: A synthetic codon-optimized gene cassette encoding tPA(sp)-PADRE-truncated ORF2 protein was designed, constructed and analyzed by some bioinformatics software. Furthermore, a codon-optimized truncated ORF2 gene was amplified by the polymerase chain reaction (PCR), with a specific primer from the previous construct. The constructs were sub-cloned in the pVAX1 expression vector and finally expressed in eukaryotic cells. RESULTS: Sequence analysis and bioinformatics studies of the codon-optimized gene cassette revealed that codon adaptation index (CAI), GC content, and frequency of optimal codon usage (Fop) value were improved, and performance of the secretory signal was confirmed. Cloning and sub-cloning of the tPA(sp)-PADRE-truncated ORF2 gene cassette and truncated ORF2 gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pVAX-tPA(sp)-PADRE-truncated ORF2 ((aa 112-660)) and pVAX-truncated ORF2 ((aa 112-660)). The expression of truncated ORF2 protein in eukaryotic cells was approved by an Immunofluorescence assay (IFA) and the reverse transcriptase polymerase chain reaction (RT-PCR) method. CONCLUSIONS: The results of this study demonstrated that the tPA(sp)-PADRE-truncated ORF2 gene cassette and the truncated ORF2 gene in recombinant plasmids are successfully expressed in eukaryotic cells. The immunogenicity of the two recombinant plasmids with different formulations will be evaluated as a novel DNA vaccine in future investigations. Kowsar 2015-12-13 /pmc/articles/PMC4744464/ /pubmed/26865938 http://dx.doi.org/10.5812/jjm.26035 Text en Copyright © 2015, Ahvaz Jundishapur University of Medical Sciences. http://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited. |
spellingShingle | Research Article Farshadpour, Fatemeh Makvandi, Manoochehr Taherkhani, Reza Design, Construction and Cloning of Truncated ORF2 and tPA(sp)-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector |
title | Design, Construction and Cloning of Truncated ORF2 and tPA(sp)-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector |
title_full | Design, Construction and Cloning of Truncated ORF2 and tPA(sp)-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector |
title_fullStr | Design, Construction and Cloning of Truncated ORF2 and tPA(sp)-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector |
title_full_unstemmed | Design, Construction and Cloning of Truncated ORF2 and tPA(sp)-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector |
title_short | Design, Construction and Cloning of Truncated ORF2 and tPA(sp)-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector |
title_sort | design, construction and cloning of truncated orf2 and tpa(sp)-padre-truncated orf2 gene cassette from hepatitis e virus in the pvax1 expression vector |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744464/ https://www.ncbi.nlm.nih.gov/pubmed/26865938 http://dx.doi.org/10.5812/jjm.26035 |
work_keys_str_mv | AT farshadpourfatemeh designconstructionandcloningoftruncatedorf2andtpasppadretruncatedorf2genecassettefromhepatitisevirusinthepvax1expressionvector AT makvandimanoochehr designconstructionandcloningoftruncatedorf2andtpasppadretruncatedorf2genecassettefromhepatitisevirusinthepvax1expressionvector AT taherkhanireza designconstructionandcloningoftruncatedorf2andtpasppadretruncatedorf2genecassettefromhepatitisevirusinthepvax1expressionvector |