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Alpha-突触核蛋白(α-synuclein,α-syn)聚集是引起帕金森病(Parkinson’s disease,PD)发生发展主要原因。本文用蛋白质/多肽片段互补分析法(protein-fragment complementation assays,PCAs)检测α-syn在细胞内的聚集。分别构建融合α-syn与人工改造的荧光素酶human Gaussiaprinceps luciferase(h GLuc)蛋白N端或C端蛋白的质粒,共转入人神经母细胞瘤SK-N-SH细胞,通过检测酶活性来确定野生型(wild type,WT)及A53T突变体α-syn在细胞中的聚集情况。结果表明WT和A53T突变α-syn都能使荧光素酶活性增强,而且与野生型α-syn相比,突变体A53T的荧光素酶活性更强,说明二者都能聚集,而且A53T聚集程度高于WT。PCAs法具有高灵敏度,不仅能检测α-syn在细胞内的聚集,而且能反映其聚集的程度,为研究帕金森病提供了研究思路和相应药物筛选的有效工具。
Alpha-synuclein (α-syn) aggregation is the main cause of the development of Parkinson’s disease (PD). In this study, protein-fragment complementation assays (PCAs) were used to detect the intracellular aggregation of α-syn. Plasmids that fused α-syn with artificial N-terminal or C-terminal protein of human Gaussia princeps luciferase (h GLuc) were co-transfected into human neuroblastoma SK-N-SH cells, respectively. The aggregation of wild type (WT) and A53T mutant α-syn in cells was determined. The results showed that both WT and A53T mutant α-syn could enhance the luciferase activity, and the luciferase activity of mutant A53T was stronger than that of wild-type α-syn, indicating that both could accumulate, and the degree of A53T accumulation Higher than WT. The high sensitivity of PCAs not only can detect the accumulation of α-syn in cells, but also reflect the degree of aggregation, which provides an effective tool for studying Parkinson’s disease and corresponding drug screening.