摘要目的 构建脑源性神经营养因子(BDNF)绿色荧光蛋白(GFP)融合表达质粒,观察其融合蛋白的特性.方法 将BDNF cDNA片段插入pcDNA3.1/NT-GFP-TOPO真核表达质粒,使其与GFP同处一个阅读框架中,测序鉴定后,转染培养的雪旺细胞,用免疫组织化学和蛋白质印迹(Western botting)观察外源性目的 蛋白的表达情况,并用该质粒活体转染视神经切断的大鼠模型,观察所表达外源性蛋白的神经保护作用.结果 测序证实质粒构建成功.Western botting结果 显示.BDNF-GFP融合蛋白表达一相对分子质量为41×103大小条带.荧光显微镜下可见BDNF-GFP转染的细胞发出绿色荧光,免疫组织化学染色后,可见绿色荧光与红色荧光完全重合.转染BDNF-GFP质粒和GFP质粒的大鼠视神经切断术后7d存活视网膜神经节细胞(RGC)分别为(1201±286)、(482±151)个/mm2,存活百分比分别为(51.39±12.24)%和(20.62±6.46)%;28 d时存活的RGC分别为(715±71)、(112±24)个/mm2,存活百分比分别为(30.59±3.04)%和(4.79±1.03)%.两组存活百分比比较,差异有统计学意义(t=3.144,11.378;P<0.01).结论 BDNF-GFP融合表达载体可以转录翻译为一融合蛋白,该蛋白可自发绿色荧光,并具有BDNF的生物学活性.

abstractsObjective 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.