论文部分内容阅读
目的:构建真核表达载体pRK5-A53T-SNCA-NLS、pRK5-A53T-SNCA-NES,瞬时转染SH-SY5Y细胞。方法:PCR扩增法获得含有SalI和NotI的目的片段A53T-SNCA-NLS、A53T-SNCA-NES,SalI、NotI双酶切载体pRK5,线性pRK5与目的片段经ligation high连接,酶切、PCR鉴定重组质粒pRK5-A53T-SNCA-NLS、pRK5-A53T-SNCA-NES。重组质粒通过Lipofactamine2000转染SH-SY5H细胞,免疫荧光法鉴定瞬转细胞。结果:真核表达载体pRK5-A53T-SNCA-NLS、pRK5-A53T-SNCA-NES构建成功,NLS、NES能介导突变基因A53T-SNCA翻译的α-synuclein蛋白在核内表达或胞质表达。结论:基因A53T-SNCA翻译的突变α-synuclein蛋白核内高表达或胞质高表达可能是家族性帕金森氏病发病机制中关键因素之一。
AIM: To construct eukaryotic expression vectors pRK5-A53T-SNCA-NLS and pRK5-A53T-SNCA-NES and transiently transfected SH-SY5Y cells. Methods: The target fragment A53T-SNCA-NLS, A53T-SNCA-NES, SalI and NotI double digested vector pRK5 containing SalI and NotI were obtained by PCR amplification. The linear pRK5 was ligated with the target fragment by ligation high, Recombinant plasmids pRK5-A53T-SNCA-NLS, pRK5-A53T-SNCA-NES. The recombinant plasmids were transfected into SH-SY5H cells by Lipofactamine 2000, and transiently transfected cells were identified by immunofluorescence. Results: The eukaryotic expression vectors pRK5-A53T-SNCA-NLS and pRK5-A53T-SNCA-NES were successfully constructed. NLS and NES mediated the expression of α-synuclein protein in nucleus or cytoplasm. CONCLUSION: High expression or high cytoplasmic expression of mutant α-synuclein protein translated by gene A53T-SNCA may be one of the key factors in the pathogenesis of familial Parkinson’s disease.