ZNF580-EGFP融合蛋白的亚细胞定位研究

Subcellular localization of ZNF580-EGFP fusion protein

目的:构建增强型绿色荧光报告蛋白(EGFP)与人ZNF580融合蛋白的真核表达载体,转染MGC803细胞进行表达,研究ZNF580-EGFP融合蛋白在MGC803细胞的亚细胞定位。方法:利用PCR技术扩增ZNF580基因cDNA开放阅读框架全编码区、N端编码区、C端编码区,分别克隆到真核表达载体pEGFP-C1,BglⅡ及HindⅢ双酶切电泳筛选、鉴定并测序。荧光显微镜下观察ZNF580-EGFP在MGC803细胞中的表达及亚细胞定位。结果:酶切pEGFP-ZNF580(1-172)、pEGFP-ZNF580(1-93)、pEGFP-ZNF580(94-172),电泳分析插入片段长度分别为:526bp、289bp、247bp,经连接点两端进行测序证实连接正确。pEGFP-ZNF580(1-172)、pEGFP-ZNF580(94-172)表达的ZNF580-EGFP融合蛋白定位在MGC803细胞核。结论:成功构建真核表达载体并在MGC803细胞中得到表达,分析其核定位信号可能位于ZNF580蛋白的C端C2H2型锌指主构域94-172位氨基酸区间。

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）.