本研究旨在对比分析猪E74 样因子 4(sELF4)和犬E74 样因子 4(cELF4)的亚细胞定位,为深入开展二者功能研究提供依据.利用PCR截短扩增sELF4 和cELF4,分别克隆至真核表达载体pEGFP-C1 中,构建 17 个sELF4 重组真核表达载体和 13 个cELF4 重组真核表达质粒,转染HeLa细胞、Hoechst33342 染色,荧光倒置显微镜观察亚细胞定位,运用生物信息学软件分析其编码氨基酸序列特征.结果表明,sELF4 和cELF4 蛋白全长均定位在细胞核中,sELF4 196~291 aa(586~873 bp)之间有可能存在两个细胞质核定位信号肽,cELF4 的核定位信号肽分布在203~291 aa(607~873 bp),cELF4 292~663 aa(874~1 992 bp)、sELF4 292~662 aa(874~1 989 bp)在细胞中发生聚集现象.cELF4 与sELF4 氨基酸序列在第 169~303 位氨基酸之间均高度保守,其他区段均存在不同程度的差异.综上,sELF4 蛋白全长和cELF4 蛋白全长均定位在细胞核中,截短表达的羧基端功能区有聚集现象,二者的核定位信号肽分布有一定差异.
This study was to compare and analyze the subcellular localization of swine E74-like factor 4(sELF4)and canine E74-like fac-tor 4(cELF4),so as to provide a basis for further functional research on the two proteins.PCR was used here to truncate sELF4 and cELF4,which were cloned into the eukaryotic expression vector pEGFP-C1,separately.17 sELF4 recombinant eukaryotic expression vectors and 13 cELF4 recombinant eukaryotic expression plasmids were constructed,transfected into HeLa cells and stained with Hoechst33342.Subcellular localization was observed by fluorescence inverted microscope,and bioinformatics software was used to analyze the sequence characteristics of encoded amino acids.The results showed that the full length of porcine and canine ELF4 proteins was localized in the nucleus.There might be two cytoplasmic nuclear localization signal peptides between sELF4 196-291aa(586-873 bp),and the nuclear localization signal peptide of cELF4 was distributed between 203-291 aa(607-873 bp).CELF4 292-663aa(874-1 992 bp)and sELF4 292-662aa(874-1 989 bp)were aggregated in the cells.The amino acid sequences of cELF4 and sELF4 were highly conserved between amino acids 169-303aa,and they were of different degrees in other parts.Taken together,canine ELF4 protein and porcine ELF4 protein were both localized in the nucle-us,and they seemed to aggregate in the truncated expression of the carboxyl terminal functional region.The distribution of nuclear localization signal peptides between them varied.