Construction, identification and application of fusion plasmid of brain-derived neurotrophic factor-green fluorescent protein

Abstract

Objective To construct expression plasmid of the fusion protein of brain-derived neurotrophic factor (BDNF)-green fluorescent protein (GFP), and observe its characteristics. Methods BDNF cDNA segment was inserted into plasmid pcDNA3.1/NT-GFP-TOPO and in the same reading frame with GFP. After verified by sequencing, the BDNF-GFP plasmid was transferred into cultured Schwann cells by electroporation. And the expression of BDNF-GFP fusion protein was observed by immunohistochemistry and Western blotting. The neural-protective function of the fusion protein was evaluated by transferring the plasmid into adult rat retinas with transected optic nerve. Results The sequence of BDNF-GFP plasmid was verified correctly by auto-sequencing. The results of Western blotting showed that the BDNF-GFP fusion protein expressed a brand with the relative molecular mass of 41×103. Seven days after the optic nerve was transected, the number of survival retinal ganglion cells (RGC) in BDNF-GFP group and GFP group was (1201±286) and (482±151) cells/mm2, respectively; and the survival rate was (51.39±12.24)% and (20.62±6.46)%, respectively. Twenty-eight days after the optic nerve was transected, the number of survival RGC in the two groups was (715±71) and (112±24) cells/mm2, respectively; the survival rate was (30.59±3.04)% and (4.79±1.03)% respectively. The differences of the survival rate of RGC between the two groups were significant (t=3. 144, 11.378; P<0.01). Conclusion BDNF-GFP fusion plasmid can express a fusion protein which emit green fluorescence and has the biological activity of BDNF. Key words: Brain-Derived neurotrophic factor/therapeutic use; Retinal ganglion cells/patho-physiology; Gene therapy; Animal experimentation