ZNF580与EGFP融合蛋白表达载体的构建及其在HEK293细胞的表达和定位

[目的]构建人ZNF580与增强型绿色荧光蛋白（EGFP）融合蛋白真核表达载体,转染细胞进行瞬时表达,分析ZNF580-EGFP融合蛋白在人胚肾HEK293细胞中的表达和亚细胞定位。[方法]利用PCR技术扩增ZNF580基因cDNA开放阅读框架全编码区（编码1-172位氨基酸）、N端编码区（编码1-93位氨基酸）、C端编码区（编码94-172位氨基酸）,分别克隆到真核表达载体pEGFP-C1,BglΠ及HindШ双酶切筛选鉴定并测序。荧光显微镜下观察ZNF580-EGFP在HEK293细胞中的表达及亚细胞定位。[结果]pEGFP-ZNF580（1-172）、pEGFP-ZNF580（94-172）表达的ZNF580-EGFP融合蛋白聚集于HEK293细胞核。[结论]ZNF580蛋白的核定位信号位于其C端C2H2型锌指主构域（94-172位氨基酸区间）。

Construction of eukaryotic expression vector for ZNF580 and EGFP fusion protein and its expression and localization in HEK293 cells

[ Objective] To constructe the eukaryotic expression plasmids for enhanced green fluorescent protein（EGFP） and ZNF580 fusion protein for investigating the subcellular localization of ZNF580-EGFP fusion protein in the transfected HEK293 cells. [Methods] The primers were designed according to the cDNA encoding sequence of ZNF580 full-length open reading frame （ 1 - 172 aa）, ZNF580 amino terminus（1 - 93 aa）and ZNF580 carboxyl terminus（94 - 172 aa）. The three cDNA segments of PCR were cloned into pGEM-T vector. They were then subcloned respectively into plasmid pEGFP-Cl（enhanced green fluorescent protein）. The subcell-ar localization of the fusion protein was monitored by autofluorescence microscopy in HEK293 cells transfected with the plasmids. Restricted enzymes analysis and DNA sequencing showed that the sequences of the recombinant plasmids pEGFP-ZNF580（1 - 172）, pEGFP-ZNF580（1 - 93） and pEGFP-ZNF580（94 - 172） were correct. [Results] The fusion proteins of pEGFP-ZNF580（1 - 172） and pEGFP- ZNF580（94 - 172） were localized in the nuclei. [Conclusions] The nuclear localization signal is within the segment between amino acid residues 94 and 172 of ZNF580 carboxyl terminus （C2H2 zinc finger domain）.