α-synuclein-pEGFP真核表达载体的构建及其在SH-SY5Y细胞中的表达

为了构建 α-synuclein-p EGFP真核表达载体 ,检测其在 SH-SY5 Y细胞内的表达 ,本研究应用下述方法 :PCR扩增 α-synuclein基因并消除终止密码子 ;PCR产物连入 p GEM T-easy载体 ,经测序确认无误后 ,亚克隆入 p EGFP-N1,构建α-synucle-in-p EGF P真核表达载体 ;Lipofect AMINE法转染 SH-SY5 Y细胞 ;荧光显微镜检测报告基因表达产物 EGFP,原位杂交和免疫荧光细胞化学法检测α-synuclein m RNA及其蛋白表达。结果显示 ,该载体转染 SH-SY5 Y细胞后 ,可在细胞内观察到报告基因和目的基因的表达产物。结论 :α-synuclein-p EGFP真核表达载体构建成功 ,并可在细胞内表达。本工作为今后动态观察研究α-synu-clein致 Parkinson病多巴胺能神经细胞损伤机制奠定基础

THE CONSTRUCTION OF α-SYNUCLEIN-pEGFP EXPRESSION VECTOR AND ITS EXPRESSION IN SH-SY5Y CELLS

To construct the mammalian expression vector of α synuclein pEGFP and detect whether it could express in SH SY5Y human dopaminergic neuroblastoma cells, the stop codons of α synuclein gene was deleted with PCR technique, then the PCR product was inserted into pGEM T easy vector. After DNA sequence analysis, α synuclein was subcloned into pEGFP N1 vector. Finally a new mammalian expression vector α synuclein pEGFP was obtained. α synuclein pEGFP was transfected with LipofectAMINE to SH SY5Y cells, and the expression of α synuclein pEGFP was determined by GFP fluorescence, in situ hybridization and anti α synuclein immunocytochemistry. The results showed that α synuclein pEGFP could express both EGFP reporter gene and α synuclein in SH SY5Y cells. α synuclein pEGFP vector was constructed successfully and it could be expressed in SH SY5Y human dopaminergic neuroblastoma cells. This work has laid foundations for further study on the roles of α synuclein in Parkinson's disease.