大黄鱼细胞适用四环素调控系统的构建与验证

doi:10.16178/j.issn.0528-9017.20231217

浙江农业科学 ›› 2024, Vol. 65 ›› Issue (6): 1285-1290.DOI: 10.16178/j.issn.0528-9017.20231217

大黄鱼细胞适用四环素调控系统的构建与验证

葛金鑫(), 颜廷鼎, 王克智, 刘也, 何志巧, 申望()

浙江海洋大学海洋科学与技术学院,浙江舟山 316022

收稿日期:2023-12-25 出版日期:2024-06-11 发布日期:2024-06-20
通讯作者: 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。
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。
基金资助:
浙江海洋大学科研启动经费(2020)

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

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

Received:2023-12-25 Online:2024-06-11 Published:2024-06-20

摘要/Abstract

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。

关键词: 四环素调控系统, 大黄鱼, 重组蛋白表达

Abstract:

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-1α promoter 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.

Key words: tetracycline-induced expression system, Larimichthys crocea, recombinant protein expression

中图分类号:

葛金鑫, 颜廷鼎, 王克智, 刘也, 何志巧, 申望. 大黄鱼细胞适用四环素调控系统的构建与验证[J]. 浙江农业科学, 2024, 65(6): 1285-1290.

GE Jinxin, YAN Tingding, WANG Kezhi, LIU Ye, HE Zhiqiao, SHEN Wang. Construction of a tetracycline-induced expression system for recombinant protein expression in large yellow croaker (Larimichthys crocea) cells[J]. Journal of Zhejiang Agricultural Sciences, 2024, 65(6): 1285-1290.

图/表 5

表1 引物信息

Table 1 Primer information

引物名称	序列(5' → 3')	用途
N1CMVF	GAATCTAAGTGGATCTGCGATCGCTCCGGTGCCCGTCAGTTAGTTATTAATAGTAATCA	载体构建
N1CMVR	TATGACTTTGCTCTTGTCCAGTCTAGACATGGTGGCGGCGAGTCCGGTAGCGCTAGC	载体构建

图1 pTRE3G-CMV载体构建流程

Fig.1 pTRE3G-CMV carrier construction process

图2 pTRE3G-CMV载体构建电泳 A—pEGFP-N1 CMV增强子/启动子PCR产物;B—泳道1: AsiSI和XcmI双酶切pTRE3G-CMV质粒,箭头示CMV增强子/ 启动子条带,泳道2:pTRE3G-CMV质粒。

Fig.2 Electrophoretic construction of pTRE3G-CMV carrier

图3 Dox诱导YCK-hk细胞pTRE3G-CMV质粒表达EGFP(100×) A—白光拍照;B—荧光拍照。

Fig.3 Expression of EGFP by pTRE3G-CMV plasmid in PCK-HK cells induced by Dox (100×)

图4 嘌呤霉素筛选pTRE3G-CMV转染细胞(200×) A—白光;B—荧光。

Fig.4 Screening of pTRE3G-CMV transfected cells with purinomycin (200×)

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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) cells

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