摘要
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).
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2008年第8期1590-1594,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金面上资助项目(No.30770885)
武警部队科研基金资助项目(No.WY2005-1
No.WJZ2007-2)