| ObjectiveConstruct the eukaryotic expression plasmids of the wild-type and A39S pathogenic mutant as well as K130R mutant DJ-1 (pEGFP-N1-DJ-1-WT, pEGFP-N1-DJ-1-A39S,pEGFP-N1-DJ-1-K130R), investigating whether DJ-1 pathogenic mutant A39S and sumoylation of DJ-1 affects mitochondria subcellular localization of DJ-1．MethodsAfter double enzyme digestion of pCDNA3．1-Myc-His(-)B-DJ-1-WT,pCDNA3．1-Myc-His(-)B-DJ-1-A39S and pGEX-5x-1-DJ-1-K130R,the aimed segments were ligased and subcloned to pEGFP-N1 vector,then the three plasmids were transfected into in vitro cultured COS-7 cells through liposome-mediated transfection,the expression of the relevant proteins were assayed by western blotting and immunofluorescence and confocal microscope.And then mitochondria staining as well as immunofluorescence were utilized to investigate whether DJ-1 pathogenic mutant A39S and sumoylation of DJ-1 play roles in mitochondria subcellular localization of DJ-1．ResultsThe correctness of the three plasmids were ascertained by double-digestion identifying and DNA sequencing.The western blotting result showed that the COS-7 cells transfected with pEGFP-N1-DJ-1-WT, pEGFP-N1-DJ-1-A39S and pEGFP-N1-DJ-1-K130R expressed successfully the target EGFP-DJ-1 fusion proteins with the molecular weight of 52KD.DJ-1-WT and DJ-1-A39S as well as DJ-1-K130R proteins showed a diffusive ubiquitous distribution in both the cytoplasm and nuclei of the transfected COS-7 cells;wild-type ,A39S pathogenic mutant DJ-1 and DJ-1-K130R proteins co-localize with mitochondria, Both DJ-1-A39S and DJ-1-K130R didn't change the subcellular localization in the mitochondria.Conclusion1．Suceedly construct the eukaryotic expression plasmids pEGFP-N1-DJ-1-WT, pEGFP-N1-DJ-1-A39S, pEGFP-N1-DJ-1-K130R. 2．DJ-1 pathogenic mutant A39S don't affect its subcellular localization to mitochondria,sumoylation of DJ-1 also don't change the mitochondria subcellular localization of DJ-1．...
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