Subcellular localization of ZNF580-EGFP fusion protein / 中国病理生理杂志

ABSTRACT

AIM:To construct a eukaryotic expression plasmid for enhanced green fluorescent protein(EGFP)and ZNF580 fusion protein,and study its subcellular localization in the transfected MGC803 cells.METHODS:The primers were designed according to the cDNA encoding sequence of ZNF580 full-length open reading frame(1-172aa),ZNF580 amino terminus(1-93aa)and ZNF580 carboxyl terminus(94-172aa).The three cDNA segments of PCR were cloned into pGEM-T vector.Then they were subcloned respectively into plasmid pEGFP-C1(enhanced green fluorescent protein).The subcellular localization of the fusion protein in MGC803 cells transfected with the vector was monitored by autofluorescence microscopy.RESULTS:Restricted enzymes analysis and DNA sequencing showed that the sequences of the pEGFP-ZNF580(1-172),pEGFP-ZNF580(1-93)and pEGFP-ZNF580(94-172)transgenic plasmid were correct.The fusion proteins of EGFP-ZNF580(1-172)and pEGFP-ZNF580(94-172)were localized in the nuclei.CONCLUSION:The recombinant eukaryotic expression vector pEGFP-ZNF580 has been successfully constructed.The nuclear localization signal is within amino acid residues 94 and 172 of ZNF580 carboxyl terminus(C2H2 zinc finger domain).