Construction of recombinant plasmid harboring APP717 mutation and preliminary study of APP proteolysis

Abstract

In order to investigate the pathogenesis of Alzheimer disease (AD) and study the enzymatic progress of amyloid precursor protein (APP), the fluorescent eukaryotic expression plasmid of C99 was constructed containing APP717 mutation. The fragment encoding the last 99-aa of APP (which was named C99 containing APP717 mutation), together with the fragment encoding yellow fluorescence protein (which was named YFP) were amplified by PCR. The two fragments (YFP and C99) were inserted into the vector pcDNA3.0. The recombinant plasmid pcDNA3.0-YFP-C99 was accomplished and its authenticity was confirmed by enzyme digestion and sequencing. Then SH-SY5Y cells were transiently transfected with the recombinant plasmid pcDNA3.0-YFP-C99. The expression of the fusion gene was detected by laser confocalmicroscopy. Amyloid-β (Aβ) was detected by both microscopy and immunochemistry. The authenticity of the construct was confirmed by the endonuclease digestion and DNA sequencing. The YFP fluorescence could be seen and proved the expression of fusion gene. Aβ labeled by YFP was detected by confocalmicroscopy and confirmed by immunocytochemistry. It was found that Aβ accumulated and deposited in the intracytoplasm, membrane and outside of the cell. Furthermore, Aβ accumulated mainly within the cell ahead of the deposition in the cell space and the cell shape was rough. It was suggested that Aβ could be generated within the cells. Aβ accumulated in the cell at the early stage before the deposition outside of the cells. Intracellular Aβ accumulation induced the secondary damage to the cells and caused the cell shape rough. Taken together, the recombinant plasmid, pcDNA3.0-YFP-C99 could be a useful tool to further study the cleavage mechanism of APP and to explore the pathogenesis of AD.

Keywords

APP / C99 / mutation / Aβ / YFP

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