SIRPα-GFP真核表达载体构建及其在HEK293T细胞中的表达

doi:10.13481/j.1671-587x.20200505

吉林大学学报(医学版) ›› 2020, Vol. 46 ›› Issue (05): 925-929.doi: 10.13481/j.1671-587x.20200505

• 基础研究 • 上一篇

SIRPα-GFP真核表达载体构建及其在HEK293T细胞中的表达

王明月, 王浩, 王冬梅, 杨泽斌, 崔梅英, 刘宁, 黄莉莉, 关新刚

北华大学医学技术学院肿瘤靶向治疗重点实验室, 吉林吉林 132013

收稿日期:2020-02-02 发布日期:2020-10-23
通讯作者: 关新刚,研究员,硕士研究生导师(Tel:0432-64608115,E-mail:guanxg@ciac.ac.cn) E-mail:guanxg@ciac.ac.cn
作者简介:王明月(1994-),女,吉林省四平市人,在读医学硕士,主要从事肿瘤免疫治疗机制方面的研究。
基金资助:
吉林省科技厅科技发展计划项目资助课题（20170520140JH，20180101213JC）；吉林省教育厅科学技术研究项目资助课题（JJKH20200033KJ）；吉林省人社厅人才开发资金项目资助课题（201858）；吉林省卫健委卫生计生青年科技骨干培养计划项目资助课题（2017Q040）；吉林省吉林市科技局科技创新发展计划项目资助课题（201831729）；北华大学青年科研创新团队项目资助课题（2017）；北华大学研究生创新计划项目资助课题（北华研创合字〔2019〕061）

Construction of SIRPα-GFP eukaryotic expression vector and its expression in HEK293T cells

WANG Mingyue, WANG Hao, WANG Dongmei, YANG Zebin, CUI Meiying, LIU Ning, HUANG Lili, GUAN Xingang

Key Laboratory of Targeting Tumor therapy, School of Medical Technology, Beihua University, Jilin 132013, China

Received:2020-02-02 Published:2020-10-23

摘要/Abstract

摘要： 目的：构建信号调节蛋白α-绿色荧光蛋白（SIRPα-GFP）真核表达载体，转染HEK293T细胞获得稳定表达SIRPα-GFP融合蛋白的细胞系，研究SIRPα-GFP融合蛋白在HEK293T细胞的膜定位。方法：将SIRPα质粒和带有GFP的表达载体分别用限制性内切酶MluⅠ和SgfⅠ进行双酶切，将SIRPα基因克隆到pLenti-GFP载体中，构建pLenti-SIRPα-GFP质粒；将pLenti-SIRPα-GFP质粒转化DH5α大肠杆菌感受态细胞，对重组质粒pLenti-SIRPα-GFP进行DNA测序；采用Lipofectamine 3000转染试剂将pLenti-SIRPα-GFP质粒转染到HEK293T细胞中，通过荧光显微镜观察SIRPα-GFP在稳定转染HEK293T细胞中的表达，采用Western blotting法检测HEK293T细胞中SIRPα-GFP融合蛋白的表达。结果：酶切鉴定和DNA测序，重组质粒pLenti-SIRPα-GFP构建成功。荧光显微镜检测，SIRPα-GFP融合蛋白在HEK293T细胞膜上表达。Western blotting法检测，SIRPα蛋白在pLenti-SIRPα-GFP质粒转染的HEK293T细胞中成功表达。结论：成功构建了pLenti-SIRPα-GFP质粒。通过在HEK293T细胞中转染pLenti-SIRPα-GFP质粒，成功制备了稳定表达SIRPα-GFP的细胞系。SIRPα-GFP蛋白定位于HEK293T细胞的细胞膜上。

关键词: 信号调节蛋白α, 绿色荧光蛋白, 真核表达载体, 细胞转染

Abstract: Objective: To construct the eukaryotic expression vector of signal regulatory protein α-green fluorescent protein (SIRPα-GFP) to transfer the HEK293T cells to establish a cell line stably expressing SIRPα-GFP fusion protein,and to investigate the membrane location of SIRPα-GFP fusion protein in the HEK293T cells. Methods: The SIRPα plasmid and the expression vector including GFP were double-digested by endonucleases MluⅠand SgfⅠ, respectively.The SIRPα gene was cloned into the pLenti-GFP vector to construct the pLenti-SIRPα-GFP plasmid.The pLenti-SIRPα-GFP plasmid was transformed into the DH5α E. coli competent cells and sequenced. The plasmids were transfected into the HEK293T cells by Lipofectamine 3000.The expression of SIRPα-GFP in the stably transfected HEK293J cells was observed by fluorescence microscope and the expression of SIRPα-GFP fusion protein in HEK293T cells was detected by Western blotting method. Results: The results of enzyme digestion and DNA sequencing showed that the recombinant plasmid pLenti-SIRPα-GFP was successfully constructed. The fluorescence microscope analysis showed that the SIRPα-GFP fusion protein was located on the membrane of HEK293T cells. The Western blotting results confirmed the successful expression of SIRPα protein in the HEK293T cells transfected with pLenti-SIRPα-GFP plasmid. Conclusion: The pLenti-SIRPα-GFP plasmid is successfully constructed. The cell line stably expressing SIRPα-GFP is successfully prepared by transfecting the pLenti-SIRPα-GFP plasmid into the HEK293T cells. The SIRPα-GFP protein is located on the membrane of HEK293T cells.

Key words: signal regulatory protein α, green fluorescent protein, eukaryotic expression vector, cell transfection

中图分类号:

R392.12

王明月, 王浩, 王冬梅, 杨泽斌, 崔梅英, 刘宁, 黄莉莉, 关新刚. SIRPα-GFP真核表达载体构建及其在HEK293T细胞中的表达[J]. 吉林大学学报(医学版), 2020, 46(05): 925-929.

WANG Mingyue, WANG Hao, WANG Dongmei, YANG Zebin, CUI Meiying, LIU Ning, HUANG Lili, GUAN Xingang. Construction of SIRPα-GFP eukaryotic expression vector and its expression in HEK293T cells[J]. Journal of Jilin University(Medicine Edition), 2020, 46(05): 925-929.

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SIRPα-GFP真核表达载体构建及其在HEK293T细胞中的表达

Construction of SIRPα-GFP eukaryotic expression vector and its expression in HEK293T cells

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