Objective To construct expression plasmid of the fusion protein of brainderived 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 BDNFGFP plasmid was transferred into cultured Schwann cells by electroporation. And the expression of BDNFGFP fusion protein was observed by immunohistochemistry and Western blotting. The neuralprotective function of the fusion protein was evaluated by transferring the plasmid into adult rat retinas with transected optic nerve.Results The sequence of BDNFGFP plasmid was verified correctly by autosequencing. The results of Western blotting showed that the BDNF-GFP fusion protein expressed a brand with the relative molecular mass of 41 times;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 plusmn;286） and（482 plusmn;151）cells/mm2, respectively; and the survival rate was （51.39 plusmn;12.24）% and （20.62 plusmn;6.46）% , respectively. Twentyeight days after the optic nerve was transected, the number of survival RGC in the two groups was （715 plusmn;71） and （112 plusmn;24）cells/mm2, respectively; the survival rate was（30.59 plusmn;3.04）% and (4.79 plusmn;1.03）% respectively. The differences of the survival rate of RGC between the two groups were significant (t=3.144，11.378；P lt;0.01).Conclusion BDNF-GFP fusion plasmid can express a fusion protein which emit green fluorescence and has the biological activity of BDNF.
Citation： 方媛,樊嘉雯,莫晓芬,孙兴怀,郭文毅,孔德升,马端,田洁. Construction, identification and application of fusion plasmid of brainderived neurotrophic factorgreen fluorescent protein. Chinese Journal of Ocular Fundus Diseases, 2009, 25(5): 372-375. doi: Copy