人野生型和A53T突变型α-突触核蛋白慢病毒表达载体的构建及在PC12细胞中的转染  

Construction of the lentivirus expression vector of wild type and A53T mutant α-synuclein and its transfection in PC12 cells

作　　者：兰丹梅[1] 邬剑军[1] 苏雅茹[1] 陈嬿[1] 蒋雨平[1] 王坚[1] 

机构地区：[1]复旦大学附属华山医院神经内科,上海200040

出　　处：《神经解剖学杂志》2009年第3期289-294,共6页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(No.30600663)资助项目

摘　　要：为构建长期稳定表达人野生型和A53T突变型α-突触核蛋白的PC12细胞株,我们首先制备了慢病毒表达载体,经测序鉴定正确后转染PC12细胞,然后通过细胞的免疫荧光染色检测α-synuclein-V5融合蛋白,PCR法扩增转基因PC12细胞的SNCA片段后进行测序检测突变基因,以及Western blot法检测PC12细胞α-synuclein的表达,从而鉴定转基因细胞能否稳定表达目的基因。结果显示:经测序,pLenti6/V5-SNCA-WT和pLenti6/V5-SNCA-A53T表达质粒构建成功;免疫荧光染色示基因转染后,超过95%的PC12细胞中有α-synuclein-V5融合蛋白表达;转基因细胞的SNCA片段测序结果显示,慢病毒表达载体成功整合入PC12细胞基因组;Western blot法检测结果示转基因PC12细胞能够过表达α-synuclein蛋白。以上结果表明我们已成功构建了人野生型和A53T突变型α-突触核蛋白慢病毒表达载体,并且成功建立了稳定的过表达人野生型和A53T突变型α-突触核蛋白的PC12细胞株,这为进一步研究α-突触核蛋白的生理功能及其在Parkinson病发病机制中的作用奠定了基础。In order to construα PC12 cells which can express wild type and A53T mutant α-synuclein for a long time, we construαed the lentivirus expression veαors and identified them by DNA sequencing at first. Then, we transfeαed the right lentivirus expression veαors into PCI2 cells. To identify whether gene transfeαed PC12 ceils can stably express objeαive gene or not, we deteαed α-synuelein-V5 merged protein by cellular immunofluorescence staining, identified mutant gene sequence by cloning SNCA gene of transfeαed PC12 cells by PCR amplification and sequencing SNCA gene, and measured the expression of α-synuclein of PC12 cells by Western blot. The results showed that the lentivirus expression veαors of pLenti6/V5-SNCA-WT and pLenti6/V5-SNCA-A53T were successfully construαed, α-synuclein-V5 merged protein was deteαed in more than 95% gene-transfeαed PC12 cells, SNCA-WT and SNCA-A53T genes were confirmed in gene-transfeαed PC12 cells by DNA sequencing,,over-expression of SNCA gene was deteαed in gene-transfeαed PC12 cells by Western blot. The above results suggest that we not only successfully construα the leutivirus expression veαors of pLenti6/V5-SNCA-WT and pLenti6/V5-SNCA-A53T, but also successfully construα PC12 cells lines stably expressing wild type and A53T mutant α-synuclein, which lays a foundation for studying its role in pathogenesis of Parkinson＇s disease in the further research.

关 键 词：Α-突触核蛋白 慢病毒表达载体 PC12细胞 PARKINSON病 转基因细胞 突变基因 

分 类 号：R346[医药卫生—基础医学]