Construction of the eukaryotic expression vector encoding IT15 gene fragment and its expression in SH-SY5Y cells / 中华神经科杂志

ABSTRACT

Objective To construct the eukaryotic expression vector encoding IT15 gene and detect its expression in SH-SY5Y cells.Methods The exon1 of IT15 gene was amplified from genomic DNA isolated from a healthy adult and a Huntington disease patient lymphoblastoid cell lines by PCR.PCR products were subcloned into eukaryotic expression vector pEGFP-C1.The SH-SY5Y cells were transiently transfected with the recombinant plasmids and observed under fluorescence microscope.Western blot was used to detect the expression of the fusion protein.Results PCR products had approximately 170 and 310 bp specific segments.The recombinant eukaryotie expression plasmids were confirmed by restriction endonuclease analysis and sequencing.The N-terminal fragment of normal huntingtin primarily localized in the cytoplasim of SH-SY5Y cells,while the N-terminal fragment of mutant huntingtin aggregated both perinuclearly and intranuelearly.Conclusions The recombinant eukaryotic expression vectors encoding wild type and mutant IT15 gene fragments are useful for studying pathogenesis of Huntington disease.The Nterminal fragment of mutant huntingtin aggregats both perinuclearly and intranuclearly,which could be important for Huntington disease pathophysiology.