摘要
构建大黄鱼(Larimichthys crocea)细胞适用的四环素调控系统,为大黄鱼蛋白质功能研究提供可靠工具。以pEGFP-N1质粒的CMV增强子/启动子替换四环素调控载体pTRE3G-MCS-EGFP-3×FLAG-Tetone-Puro(简称pTRE3G)转录激活子(rtTA)的EF-1α启动子及其下游5′-长末端重复序列(5′-LTR),构建重组载体pTRE3G-CMV;转染大黄鱼头肾细胞系YCK-hk细胞,Dox诱导EGFP表达,验证pTRE3G-CMV在大黄鱼细胞中驱动外源基因表达的可靠性。成功构建四环素调控载体pTRE3G-CMV;pTRE3G-CMV质粒转染的大黄鱼YCK-hk细胞经Dox诱导表达EGFP,表明CMV增强子/启动子在YCK-hk细胞中正常驱动rtTA表达;pTRE3G-CMV质粒转染YCK-hk细胞效率低(<1%),经嘌呤霉素筛选后EGFP表达阳性细胞率上升至约30%,表明嘌呤霉素能有效筛选转染成功细胞,提示可结合单克隆筛选建立稳转细胞系。研究构建的四环素调控载体pTRE3G-CMV在大黄鱼细胞中正常表达外源基因,可用于大黄鱼蛋白质功能研究。
In order to construct a tetracycline-induced expression system for recombinant protein expression in large yellow croaker(Larimichthys crocea)cells to study the protein function of large yellow croaker,a Tet-On system recombinant plasmid,designated as pTRE3G-CMV,was constructed by replacing the EF-1αpromoter and its downstream 5′-long terminal repeat(5′-LTR)sequence of transcription activator(rtTA)of a Tet-On 3G inducible expression plasmid pTRE3G-MCS-EGFP-3×FLAG-Tetone-Puro with the CMV enhancer/promoter of pEGFP-N1 plasmid.When transfected YCK-hk(large yellow croaker head kidney cell line)cells with pTRE3G-CMV plasmid DNA by lipofection reagent,the expression of EGFP positive cells was observed post doxycycline induction for 3 h,but the percentages of EGFP positive cells was low(<1%).After screened with purinomycin,the EGFP positive ratio of cells can be increased to about 30%,suggesting that the pTRE3G-CMV stable cell lines can be established by combined with purinomycin screening technique and single-cell clonal lines screening technique.These results indicated that the Tet-On plasmid pTRE3G-CMV constructed in this study can normally express foreign genes in large yellow croaker cells and can be used to study the protein function of large yellow croaker in vitro.
作者
葛金鑫
颜廷鼎
王克智
刘也
何志巧
申望
GE Jinxin;YAN Tingding;WANG Kezhi;LIU Ye;HE Zhiqiao;SHEN Wang(Laboratory of Marine Biology Resource and Molecular Engineering,Marine Science and Technical College,Zhejiang Ocean University,Zhoushan 316022,Zhejiang)
出处
《浙江农业科学》
2024年第6期1285-1290,共6页
Journal of Zhejiang Agricultural Sciences
基金
浙江海洋大学科研启动经费(2020)。
关键词
四环素调控系统
大黄鱼
重组蛋白表达
tetracycline-induced expression system
Larimichthys crocea
recombinant protein expression
作者简介
葛金鑫(2002-),本科生,主要从事鱼类养殖病害防治等研究工作,E-mail:2740812532@qq.com。;通信作者:申望(1975-),副教授,博士,主要从事水产养殖病害防治等研究工作,E-mail:shenwang@zjou.edu.cn。
