title: Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3.
abstract: 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.
authors.affiliation: Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Key Laboratory of Ophthalmology & Visual Sciences, Beijing 100730, China.
authors.forename: Chunxia
authors.initials: C
authors.lastname: Peng
authors.affiliation: Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Key Laboratory of Ophthalmology & Visual Sciences, Beijing 100730, China.
authors.forename: Xiaobei
authors.initials: X
authors.lastname: Yin
authors.affiliation: Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Key Laboratory of Ophthalmology & Visual Sciences, Beijing 100730, China.
authors.forename: Mengda
authors.initials: M
authors.lastname: Li
authors.affiliation: Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Key Laboratory of Ophthalmology & Visual Sciences, Beijing 100730, China.
authors.forename: Ting
authors.initials: T
authors.lastname: He
authors.affiliation: Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Key Laboratory of Ophthalmology & Visual Sciences, Beijing 100730, China.
authors.forename: Genlin
authors.initials: G
authors.lastname: Li
keywords: biological factor; encapsulated cell technology; fusion protein; gene therapy; grants-supported paper; neural regeneration; neuroregeneration; neurotrophin-3; plasmid; retinitis pigmentosa