红鳍东方鲀PPARγ真核基因表达载体构建及亚细胞定位研究

Construction of Eukaryotic Expression Vector and Subcellular Localization of PPARγ in torafugu Takifugu rubripes

[目的]旨在克隆红鳍东方鲀PPARγ基因的cDNA,并通过构建真核表达载体pCMV-3Tag-4A-PPARγ,分析myc-PPARγ融合蛋白在大鼠L6细胞的表达及亚细胞定位。[方法]利用RT-PCR技术克隆红鳍东方鲀PPARγ基因的cDNA,构建pCMV-3Tag-4A-PPARγ重组质粒,转染至大鼠L6细胞,共聚焦显微镜下观察myc-PPARγ的亚细胞定位。[结果]红鳍东方鲀PPARγ基因cDNA ORF全长1557 bp,共编码518个氨基酸,pCMV-3Tag-4A-PPARγ融合蛋白在大鼠L6细胞核中表达,为深入探讨鱼类PPARγ生物学功能奠定了基础。

[Objective] The aim of the study was to clone eDNA ofPPA RT in Tokifugu rubripes,and analyzed subcellular locahzation of the expression product through myc fusion protein.[Method] The eDNA of Takifugu rubripes PPARγ gene was cloned by RT-PCR and the recombinant eukaryotic expression vectors pCMV-3Tag-4A-PPARγ were constructed and used for rat L6 ceils transfection.[Result] The eDNA ORF of PPA RT had a length of 1 557 bp encoding 518 amino acids.The polyclonal antibody against PPARγ had been prepared successfully;the conjugated protein was emciently expressed inthe cytoplasm of rat L6 cells.These results had a good value for further research of PPA RT function.