团头鲂oct4基因真核表达载体的构建与表达  

Construction and expression of eukaryotic expression vector of oct4 gene in Megalobrama amblycephala

作　　者：于淼 潘启华 王乾[2] 方健[2] 薛亭 陈天圣[2,3,4,5] YU Miao;PAN Qihua;WANG Qian;FANG Jian;XUE Ting;CHEN Tiansheng(College of Fisheries,Henan Normal University,Xinxiang 453007,Henan,China;College of Fisheries,Huazhong Agricultural University,Wuhan 430070,Hubei,China;Key Laboratory of Freshwater Animal Breeding,Ministry of Agriculture and Rural Affairs,Wuhan 430070,Hubei,China;Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan Province,Changde 415000,Hunan,China;Fisheries College of Jimei University,Xiamen 361021,Fujian,China)

机构地区：[1]河南师范大学水产学院,河南新乡453007 [2]华中农业大学水产学院,湖北武汉430070 [3]农业农村部淡水生物繁育重点实验室,湖北武汉430070 [4]水产高效健康生产湖南省协同创新中心,湖南常德415000 [5]集美大学水产学院,福建厦门361021

出　　处：《上海海洋大学学报》2021年第3期399-406,共8页Journal of Shanghai Ocean University

基　　金：国家自然科学基金(31672653,31771648);华中农业大学科技自主创新基金(2013RC014,2662015PY049);河南省科技攻关项目(192102110083);天津市水产生态及养殖重点实验室开放基金(TJAE201806);河南师范大学博士科研启动基金(QD18095)。

摘　　要：八聚体结合转录因子4(octamer-binding transcription factor 4,Oct4)在干细胞多能性维持、胚胎早期发育和体细胞重编程等过程中具有重要作用,构建团头鲂oct4基因真核表达载体可为进一步研究该基因的功能提供实用工具。通过PCR方法从含有团头鲂oct4基因开放阅读框序列的载体pCS2+Maoct4中获得基因编码片段,连入真核表达载体pCVpr,构建重组的融合表达载体pCMV-Maoct4-Red;经酶切和测序鉴定后,采用脂质体法将该载体转染HepG2细胞,观察到细胞中红色荧光主要定位于细胞核,并采用Western blot方法分析融合蛋白的正确表达。成功构建了团头鲂oct4基因荧光真核表达载体pCMV-Maoct4-Red,该载体可用于真核细胞的核定位标记,为后续研究鱼类oct4基因的作用机制奠定了基础。The transcription factor oct4 plays an important role in maintaining the pluripotency of stem cells,early embryonic development and reprogramming of somatic cells.Construction of the eukaryotic expression vector of oct4 gene from Megalobrama amblycephala(Maoct4)can provide a practical tool for further study on the mechanism of the gene.The target gene fragment was obtained from the pCS2+Maoct4 vector containing the ORF sequence of the Maoct4 gene,and was cloned into pCVpr to construct the infusion and recombinant vector of pCMV-Maoct4-Red.After enzyme digestion and sequencing identification,the recombinant vector was transfected into HepG2 cells by liposome,and the expression and localization of the red fluorescence in HepG2 cells were observed in the nucleus,and the fusion protein was also detected by Western blot using MaOct4 antibody.Therefore,the fluorescent eukaryotic expression vector of pCMV-Maoct4-Red was successfully constructed,which could be effectively localized in the nucleus in eukaryotic cells,which would lay the foundation for further study on the function of the Maoct4 gene.

关 键 词：团头鲂 OCT4 真核表达载体 亚细胞定位 

分 类 号：S917.4[农业科学—水产科学]