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An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons
Published on: July 11, 2020
Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial
Bo-Jing Yan1, Zhi-Zhong Wu1, Wei-Hua Chong1
1Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Sciences Key Lab, Beijing, China.
Abstract:
Several studies have investigated the protective functions of brain-derived neurotrophic factor (BDNF) in retinitis pigmentosa. However, a BDNF-based therapy for retinitis pigmentosa is not yet available. To develop an efficient treatment for fundus disease, an eukaryotic expression plasmid was generated and used to transfect human 293T cells to assess the expression and bioactivity of BDNF on acute retinal pigment epithelial-19 (ARPE-19) cells, a human retinal epithelial cell line. After 96 hours of co-culture in a Transwell chamber, ARPE-19 cells exposed to BDNF secreted by 293T cells were more viable than ARPE-19 cells not exposed to secreted BDNF. Western blot assay showed that Bax levels were downregulated and that Bcl-2 levels were upregulated in human ARPE-19 cells exposed to BDNF. Furthermore, 293T cells transfected with the BDNF gene steadily secreted the protein. The powerful anti-apoptotic function of this BDNF may be useful for the treatment of retinitis pigmentosa and other retinal degenerative diseases.
