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

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]