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Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3☆
Neurotrophin-3 (NT-3) can promote the repair of central nervous system and retinal damage. In previous reports, NT-3 has been expressed by viral vectors. However, plasmid vectors have a safer profile compared with viral vectors in clinical studies. This study recombined amplified human retinal NT-3...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Medknow Publications & Media Pvt Ltd
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4145883/ https://www.ncbi.nlm.nih.gov/pubmed/25206397 http://dx.doi.org/10.3969/j.issn.1673-5374.2013.11.009 |
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author | Peng, Chunxia Yin, Xiaobei Li, Mengda He, Ting Li, Genlin |
author_facet | Peng, Chunxia Yin, Xiaobei Li, Mengda He, Ting Li, Genlin |
author_sort | Peng, Chunxia |
collection | PubMed |
description | Neurotrophin-3 (NT-3) can promote the repair of central nervous system and retinal damage. In previous reports, NT-3 has been expressed by viral vectors. However, plasmid vectors have a safer profile compared with viral vectors in clinical studies. This study recombined amplified human retinal NT-3 with a eukaryotic expression plasmid containing green fluorescent protein (GFP) to construct an NT-3 expression plasmid, pEGFP-N1-NT-3. The transfection efficiency 48 hours after pEGFP-N1-NT-3 transfection to 293T cells was 50.06 ± 2.78%. Abundant NT-3-GFP was expressed in 293T cells as observed by fluorescence microscopy, suggesting the construct pEGFP-N1-NT-3 effectively expressed and secreted NT-3-GFP. Secretory vesicles containing NT-3-GFP were observed in a constant location in cells by laser scan confocal microscopy, indicating the expression and secretion processes of NT-3 in eukaryotic cells were in accordance with the physical synthesis processes of secreted proteins. Western blot assay showed that pro-NT-3-GFP had a molecular weight of 56 kDa, further confirming NT-3-GFP expression. At 48 hours after transfection, the concentration of NT-3 in culture medium was 22.3 ng/mL, suggesting NT-3 produced by pEGFP-N1-NT-3 was efficiently secreted. This study constructed a human retinal-derived NT-3 eukaryotic expression plasmid that efficiently expressed and secreted NT-3. |
format | Online Article Text |
id | pubmed-4145883 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Medknow Publications & Media Pvt Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-41458832014-09-09 Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3☆ Peng, Chunxia Yin, Xiaobei Li, Mengda He, Ting Li, Genlin Neural Regen Res Neurodegenerative Disease and Neural Regeneration Neurotrophin-3 (NT-3) can promote the repair of central nervous system and retinal damage. In previous reports, NT-3 has been expressed by viral vectors. However, plasmid vectors have a safer profile compared with viral vectors in clinical studies. This study recombined amplified human retinal NT-3 with a eukaryotic expression plasmid containing green fluorescent protein (GFP) to construct an NT-3 expression plasmid, pEGFP-N1-NT-3. The transfection efficiency 48 hours after pEGFP-N1-NT-3 transfection to 293T cells was 50.06 ± 2.78%. Abundant NT-3-GFP was expressed in 293T cells as observed by fluorescence microscopy, suggesting the construct pEGFP-N1-NT-3 effectively expressed and secreted NT-3-GFP. Secretory vesicles containing NT-3-GFP were observed in a constant location in cells by laser scan confocal microscopy, indicating the expression and secretion processes of NT-3 in eukaryotic cells were in accordance with the physical synthesis processes of secreted proteins. Western blot assay showed that pro-NT-3-GFP had a molecular weight of 56 kDa, further confirming NT-3-GFP expression. At 48 hours after transfection, the concentration of NT-3 in culture medium was 22.3 ng/mL, suggesting NT-3 produced by pEGFP-N1-NT-3 was efficiently secreted. This study constructed a human retinal-derived NT-3 eukaryotic expression plasmid that efficiently expressed and secreted NT-3. Medknow Publications & Media Pvt Ltd 2013-04-15 /pmc/articles/PMC4145883/ /pubmed/25206397 http://dx.doi.org/10.3969/j.issn.1673-5374.2013.11.009 Text en Copyright: © Neural Regeneration Research http://creativecommons.org/licenses/by-nc-sa/3.0 This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Neurodegenerative Disease and Neural Regeneration Peng, Chunxia Yin, Xiaobei Li, Mengda He, Ting Li, Genlin Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3☆ |
title | Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3☆ |
title_full | Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3☆ |
title_fullStr | Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3☆ |
title_full_unstemmed | Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3☆ |
title_short | Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3☆ |
title_sort | construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3☆ |
topic | Neurodegenerative Disease and Neural Regeneration |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4145883/ https://www.ncbi.nlm.nih.gov/pubmed/25206397 http://dx.doi.org/10.3969/j.issn.1673-5374.2013.11.009 |
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