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...

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Vydáno v:Neural regeneration research Ročník 8; číslo 11; s. 1031 - 1040
Hlavní autoři: Peng, Chunxia, Yin, Xiaobei, Li, Mengda, He, Ting, Li, Genlin
Médium: Journal Article
Jazyk:angličtina
Vydáno: India Medknow Publications and Media Pvt. Ltd 15.04.2013
Beijing Tongren Eye Center,Beijing Tongren Hospital, Capital Medical University, Beijing Key Laboratory of Ophthalmology & Visual Sciences, Beijing 100730, China
Medknow Publications & Media Pvt Ltd
Témata:
Bioengineering
Methods
Neurodegenerative Disease and Neural Regeneration
Neurotrophic functions
Physiological aspects
Plasmids
Retina
encapsulated cell technology
fusion protein
neural regeneration
neuroregeneration
grants-supported paper
plasmid
retinitis pigmentosa
gene therapy
biological factor
neurotrophin-3
ISSN:1673-5374, 1876-7958
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Shrnutí: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.
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Author statements: The manuscript is original, has not been submitted to or is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential information or authorship/patent application/funding source disputations.
Author contributions: Chunxia Peng performed the majority of the experiments, wrote the manuscript, provided data, and performed data analysis. Xiaobei Yin conducted part of the experiments. Mengda Li and Ting He participated in the study instruction and analysis. Genlin Li was the study designer, the paper validator, and the fund header. All authors have read and approved the final version of the manuscript.
Chunxia Peng☆, Studying for doctorate.
ISSN:1673-5374
1876-7958
DOI:10.3969/j.issn.1673-5374.2013.11.009