pEGFP-N1-GGF2质粒的构建及其在大鼠脑内的表达

Construction of the plasmid pEGFP-N1-GGF2 and its expression in the rat brain

目的构建pEGFP-N1-GGF2质粒,观察其在大鼠脑组织中的表达。方法采用RT-PCR方法从人胚胎脑组织总RNA中扩增出人GGF2全长序列,并克隆到增强型绿色荧光蛋白(EGFP)基因真核表达载体pEGFP-N1上,构建pEGFP-N1-GGF2质粒,通过脂质体将pEGFP-N1-GGF2质粒转染大鼠脑组织,荧光显微镜下观察GGF2融合蛋白在大鼠脑内表达的时间和空间分布特征。结果成功构建了pEGFP-N1-GGF2表达载体,荧光显微镜观察可见,pEGFP-N1-GGF2表达载体转染6 h大鼠脑内即可见GGF2融合蛋白表达,3 d时融合蛋白表达最多,7 d时表达量稍有下降但仍高,14 d时表达量已明显下降。GGF2融合蛋白在脑内的表达以针道为中心向周围扩散,荧光信号可达皮层深部。结论脂质体介导的pEGFP-N1-GGF2质粒能够在大鼠脑内表达GGF2融合蛋白。

Objective To construct the plasmid pEGFP-N 1-GGF2 and observe its expression in the rat brain. Methods Full-length sequences of GGF2 were amplified from total RNA of the human fetal brain tissue by reverse transcription-polymerase chain reaction （RT-PCR） and cloned into the eukaryotic expression vector pEGFP-N1 encoding the enhanced green fluorescent protein （EGFP） gene. The plasmid pEGFP-N1-GGF2 was constructed and transfected into the rat brain by liposome. Temporal and spatial distributions of GGF2 fusion protein expressed in the rat brain were observed under a fluorescence microscope. Results The glial growth factor eukaryotic expression vector pEGFP-N1-GGF2 was successfully constructed. The expression of GGF2 fusion protein was detected in the rat brain at 6 h after transfection, rose to its peak at 3 d, decreased slightly at 7 d though still high and decreased evidently at 14 d. GGF2 fusion protein existed along the injection track and diffused around in the rat brain; some fluorescent signals might reach deep cortex. Conclusion The liposome-mediated plasmid pEGFP-N1-GGF2 may express GGF2 fusion protein in the rat brain.