摘要
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 sELF4196-291aa(586-873 bp),and the nuclear localization signal peptide of cELF4 was distributed between 203-291 aa(607-873 bp).CELF4292-663aa(874-1992 bp)and sELF4292-662aa(874-1989 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.
作者
高翠翠
喻娇
唐青海
侯鑫军
刘婷
全飞杨
刘建行
赵婷芳
赵铖
朱钰英
危艳武
GAO Cuicui;YU Jiao;TANG Qinghai;HOU Xinjun;LIU Ting;QUAN Feiyang;LIU Jianxing;ZHAO Tingfang;ZHAO Cheng;ZHU Yuying;WEI Yanwu(Hunan Key Laboratory of Conservation and Utilization of Biological Resources in the Nanyue Mountainous Region/Hengyang Key Laboratory of New Detection Technology and Biological Agents of Animal Microorganism,College of Life Sciences,Hengyang Normal University,Hengyang 421008,China;State Key Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《畜牧与兽医》
CAS
北大核心
2024年第3期45-53,共9页
Animal Husbandry & Veterinary Medicine
基金
国家级大学创新创业训练项目(cxcy2022001)
湖南省自然科学基金面上项目(2021JJ30060)
湖南省教育厅科学研究项目重点项目(21A0442)
湖南省重点研发计划项目(2021NK2026)
2022年中央引导地方科技发展资金项目(2022ZYC091)。
关键词
猪ELF4蛋白
犬ELF4蛋白
真核表达
亚细胞定位
swine ELF4 protein
canine ELF4 protein
eukaryotic expression
subcellular localization
