SNCA基因过表达慢病毒质粒构建及稳定转染293T细胞系

Stably expression human SNCA gene in 293T cells by Lentivirus

目的构建SNCA基因过表达慢病毒质粒,转染293T细胞,建立稳定转染细胞系。方法应用PCR技术扩增目的基因,并将扩增产物插入慢病毒载体质粒pGC-FU上,并对阳性克隆进行基因测序鉴定。pGC-FU-SNCA-GFP重组质粒包装293T细胞,转染24小时后,用荧光显微镜观察标签GFP绿色荧光蛋白的表达,并用West blotting法测定目的蛋白的表达。结果成功构建了pGC-FU-SNCA-GFP慢病毒过表达质粒,获得了稳定转染的293T细胞株。结论人SNCA基因过表达慢病毒载体成功,构建和稳定转染293T细胞系的建立,为进一步体外研究α-突触核蛋白的功能奠定了基础。

Objective Construction and identification of human SNCA gene lentiviral expression vector,and its stable transfection into the 293T cells.Methods SNCA was synthesized with particular primer,amplified by PCR and cloned into the lentiviral vector expression plasmid pGC-FU.After digestion and sequencing,pGC-FU-SNCA-GFP was co-transfected into 293T cells.Result By detecting the level of tagged protein of green fluorescent protein(GFP) and the target protein,the pGC-FU-SNCA-GFP expression in target cells was verified.Conclusion The human SNCA proteins are stably expressed in the 293T cells,for further the basal function of a-synuclein.