Construction of the eukaryotic expression vector of MJD1 and its expression in SH-SY5Y cells / 中南大学学报(医学版)
Journal of Central South University(Medical Sciences)
; (12): 640-644, 2005.
Article
Dans Zh
| WPRIM
| ID: wpr-813458
Responsable en Bibliothèque :
WPRO
ABSTRACT
OBJECTIVE@#To construct the eukaryotic expression vector of MJD1 with normal copies of CAG trinucleotide repetition and MJD1 with CAG trinucleotide repetition expansion mutation respectively, and to determine whether the polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation.@*METHODS@#The coding sequence of wild-type MJD1 and mutant MJD1 was amplified by PCR from pAS2-1-MJD20Q and pAS2-1-MJD68Q respectively. After being digested with BamH I and Hind III, the PCR products were inserted into pcDNA3. 1-Myc-His(-) B. The recombinant plasmids pcDNA3.1-Myc-His(-) B-MJD20Q and pcDNA3.1-Myc-His(-) B-MJD68Q were identified by enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into SH-SYSY cells and the expression of MJD1 in the transfected cells was analyzed by Western blot. The immunofluorescence of the transfected cells was examined using a confocal microscope to observe the formation of intranuclear aggregation.@*RESULTS@#Enzyme digestion analysis and DNA sequencing showed that the target gene was cloned into pcDNA3. 1-Myc-His(-) B. The expression of MJD1 in the transfected cells was confirmed by Western blot; The SH-SY5Y cells transfected with pcDNA3. 1-Myc-His(-) B-MJD68Q showed the formation of intranuclear aggregation, but the cells transfected with pcDNA3.1-Myc-His(-) B-MJD20Q did not show such phenomenon.@*CONCLUSION@#The eukaryotic expression vectors of MJD1 has been successfully constructed; The polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation.
Texte intégral:
1
Indice:
WPRIM
Sujet Principal:
Anatomopathologie
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Plasmides
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Protéines de répression
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Récepteurs des hormones thyroïdiennes
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Protéines recombinantes
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Données de séquences moléculaires
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Protéines nucléaires
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Cellules cancéreuses en culture
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Séquence nucléotidique
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Transfection
Type d'étude:
Prognostic_studies
Limites du sujet:
Humans
langue:
Zh
Texte intégral:
Journal of Central South University(Medical Sciences)
Année:
2005
Type:
Article