斜带石斑鱼PKR基因的克隆表达及亚细胞定位分析

Molecular Cloning,Expression and Subcellular Localization of PKR from Epinephelus coioides

根据实验室前期获得的斜带石斑鱼PKR基因的全长c DNA,对其原核表达、组织分布及亚细胞定位进行了研究分析。结果表明,斜带石斑鱼PKR基因c DNA全长为2 151 bp,其中ORF区为1 863 bp,编码621个氨基酸,表达蛋白的分子质量大小为70 157.15 Da,PI为5.72。通过NCBI网站上BLAST软件比对发现,斜带石斑鱼PKR与其他物种的PKR高度同源。通过构建原核表达载体PKR-p ET-32a,获得了斜带石斑鱼PKR的融合蛋白,经纯化后制备了斜带石斑鱼PKR鼠抗血清。利用荧光定量PCR对斜带石斑鱼PKR基因组织分布研究表明,该基因在所有检测组织中均有表达,其中肠的表达量最高,头肾次之。以荧光显微镜对其亚细胞定位进行了分析,该基因为全细胞分布,但主要分布于细胞质中。

The full length c DNA of PKR gene was cloned from the liver of grouper,Epinephelus coioides,which is 2 151 bp,including 1 863 bp ORF area,encoding 621 amino acids. Its protein molecular weight is 70 157.15 Daltons and PI is 5. 72. BLAST comparison on the NCBI web site found that the PKR of the grouper was highly homologous to that of other species. By constructing prokaryotic expression vector PKR-p ET-32 a,the fusion protein of PKR was obtained and purified. The mouse antiserum of PKR was also prepared. Real-time PCR analysis indicates that the PKR gene is expressed in all tissues of the grouper,most highly in the intestine and then in the head kidney. Subcellular localization of PKR showed that PKR was distributed in both nucleus and cytoplasm,but mainly in the cytoplasm.