大黄鱼细胞适用四环素调控系统的构建与验证  

Construction of a tetracycline-induced expression system for recombinant protein expression in large yellow croaker(Larimichthys crocea)cell

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)

机构地区：[1]浙江海洋大学海洋科学与技术学院,浙江舟山316022

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)。

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.

关 键 词：四环素调控系统 大黄鱼 重组蛋白表达 

分 类 号：S917.4[农业科学—水产科学] Q952.6[生物学—动物学]