脑源性神经营养因子-绿色荧光蛋白融合基因载体的构建、鉴定及初步应用研究  

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

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作  者:方媛[1] 樊嘉雯[1] 莫晓芬[1] 孙兴怀[1] 郭文毅[1] 孔德升[2] 马端[2] 田洁[1] 

机构地区:[1]复旦大学附属眼耳鼻喉科医院,上海200031 [2]复旦大学分子医学教育部重点实验室

出  处:《中华眼底病杂志》2009年第5期372-375,共4页Chinese Journal of Ocular Fundus Diseases

基  金:基金项目:国家自然科学基金(30300379,30672281);上海市自然科学基金(034119807);国家重点基础研究发展计划(2007CB512204);教育部新世纪优秀人才计划(NCET-050370);复旦大学脑科学开放基金、上海医学院交叉基金、医学神经生物学重点实验室开放课题(09-07)

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BDNFcDNA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。Westernbotting结果显示,BDNF—GFP融合蛋白表达一相对分子质量为41×10^3。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/mm^2,存活百分比分别为(51.39±12.24)%和(20.62±6.46)%;28d时存活的RGC分别为(715±71)、(112±24)个/mm^2,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。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× 10^3. Seven clays after the optic nerve was transected, the number of survival retinal ganglion ceils (RGC) in BDNF-GFP group and GFP group was (1201± 286) and(482 ±151 )cells/mm^2, 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/mm^2 , 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.

关 键 词:脑源性神经营养因子/治疗应用 视网膜神经节细胞/病理生理学 基因疗法 动物实验 

分 类 号:R774.1[医药卫生—眼科] R346[医药卫生—临床医学]

 

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