解剖学报

解剖学报 ›› 2018, Vol. 49 ›› Issue (5): 591-597.doi: 10.16098/j.issn.0529-1356.2018.05.005

• 细胞和分子生物学 • 上一篇    下一篇

稳定表达α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体GluA1亚基单克隆细胞株的构建

李炯1 张吉凤1 赵波1,2蔡振彬1,3朱肖楠1 郭国庆1*   

  1. 1. 暨南大学基础医学院解剖学系，广州 510632; 2. 暨南大学附属第一医院康复科，广州 510630; 3. 暨南大学附属第一医院骨科， 广州 510630
  • 收稿日期:2017-09-21 修回日期:2018-04-03 出版日期:2018-10-06 发布日期:2018-10-06
  • 通讯作者: 郭国庆 E-mail:tgqguo@jnu.edu.cn
  • 基金资助:
    国家自然科学基金项目;国家自然科学基金项目;广东省自然科学基金重点项目

Establishment of monoclonal cell strain stably expressing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluA1

LI Jiong1 ZHANG Ji-feng1 ZHAO Bo 1,2 CAI Zhen-bin 1,3 ZHU Xiao-nan1 GUO Guo-qing 1*   

  1. 1 Department of Anatomy, Medical College of Ji’nan University, Guangzhou 510632, China; 2. Department of Rehabilitation, the First Affiliated Hospital of Ji’nan University, Guangzhou 510632, China;  3. Department of Orthopedics, the First Affiliated Hospital of Ji’nan University, Guangzhou 510632, China
  • Received:2017-09-21 Revised:2018-04-03 Online:2018-10-06 Published:2018-10-06
  • Contact: GUO Guo-qing E-mail:tgqguo@jnu.edu.cn

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摘要：

目的 构建稳定表达α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体GluA1亚基的细胞株。 方法 PCR扩增GluA1目的基因，并将其装载至慢病毒表达载体PLV.0-绿色荧光蛋白（GFP），采用4质粒系统转染HEK293T细胞包装慢病毒。嘌呤霉素抗性筛选细胞，挑选单克隆细胞株用Real-time PCR、Western blotting、免疫荧光和全细胞膜片钳鉴定GluA1的表达与功能。 结果 菌落PCR鉴定扩增产物与GluA1分子量（2724 bp）一致，经测序插入慢病毒载体序列与GluA1序列一致；慢病毒感染并抗性筛选的HEK293T细胞经Real-time PCR、Western blotting和免疫荧光实验证明，GluA1在过表达GluA1组的HEK293T细胞正确表达，空载体组不表达。单克隆挑选的稳定细胞株免疫荧光染色显示，各细胞膜表面GluA1蛋白表达相对一致。单克隆细胞在电压钳模式下，细胞外液加10 mmol/L谷氨酸诱导，空载体组检测不到电信号，而过表达GluA1组可记录5~40 pA电信号。 结论 成功构建了稳定表达AMPA受体GluA1亚基的HEK293T细胞株。

关键词: GluA1亚基, 慢病毒, 单克隆, 细胞株, 实时定量聚合酶链反应, 免疫印迹法

Abstract:

Objective To build a cell line stably expressing α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid(AMPA) receptor GluA1 subunit. Methods The GluA1 gene was amplified by PCR and loaded into the lentiviral expression vector PLV.0-green fluorescent protein(GFP). HEK293T cells were transfected by using the four plasmid system. Puromycin was used for screening stable cell lines. The monoclonal cells were identified by Real-time PCR, Western blotting, immunofluorescence and whole cell patch clamp. Results The amplified product identified by colony PCR was consistent with the molecular weight of GluA1 (2724 bp). Further sequencing result showed that the sequence inserted into the lentiviral vector was consistent with the GluA1 sequence; HEK293T stable cell lines screened by puromycin selection were confirmed by Real-time PCR, Western blotting and immunofluorescence. GluA1 expression was correct in the experimental group, but not expressed in the control group. Immunofluorescent staining showed that the expression of GluA1 protein was relatively uniform on each cell surface. Stable cell lines were detected in voltage clamp mode with 10 mmol/L glutamate in extracellular fluid, and no electrical signal was detected in the empty vector group, whereas 5-40 pA electrical signal was recorded in the GluA1 overexpressing group. Conclusion We successfully established a HEK293T monoclonal stable cell strain expressing GluA1 subunit.

Key words: GluA1 subunit, Lentiviral, Monoclonal, Cell strain, Real-time PCR, Western blotting