贵州医科大学学报

2014, v.39;No.171(06) 794-799

[打印本页] [关闭]
本期目录(Current Issue) | 过刊浏览(Past Issue) | 高级检索(Advanced Search)

Gi蛋白真核表达载体的构建及表达检测
Construction and Detection of the Eukaryotic Expression Vectors of Gi Protein Gene

胡祖权;何天军;贾义;宋萍萍;邱炜;赵远波;曾柱;
HU Zuquan;HE Tianjun;JIA Yi;SONG Pingping;QIU Wei;ZHAO Yuanbo;ZENG Zhu;Department of Biotechnology,College of Biology and Engineering,Guiyang Medical University;

摘要(Abstract):

目的:克隆抑制腺苷酸环化酶G蛋白(Gi蛋白)3个亚基α、β和γ基因,构建p EYFP-N-Giα1和p ECFP-C-Giβ1-γ2真核表达载体,完成其在真核细胞中的表达检测。方法:培养肝癌细胞Hep G2,提取总RNA后反转录成c DNA,通过聚合酶链式反应(PCR)扩增α、β和γ的编码基因,在α亚基两端添加酶切位点,在β和γ亚基两端分别添加酶切位点和连接肽,经酶切和连接得到Giα1和Giβ1-γ2,再将Giα1和Giβ1-γ2基因分别构建到荧光蛋白报告载体p EYFP-N1和p ECFP-C1中,利用脂质体法将真核表达载体转染到肝癌细胞中,激光共聚焦显微镜观察分析其表达活性。结果:PCR扩增获得Gi蛋白α、β和γ亚基的编码基因,在两端添加酶切位点和连接肽后获得的Giα1和Giβ1-γ2基因,成功构建了p EYFP-N-Giα1和p ECFP-C-Giβ1-γ2真核表达载体,并在肝癌细胞中实现其真核细胞表达。结论:成功构建Gi蛋白的真核表达载体,并鉴定其在活细胞中具有表达活性。
Objective: To isolate the alpha,beta and gamma subunit genes of Gi protein and construct the eukaryotic expression vectors of p EYFP-N-Giα1and p ECFP-C-Giβ1-γ2; detecting the expression of vectors in the eukaryotic cells. Methods: Hep G2 liver cancer cells were cultured and the total RNA was isolated. Then c DNA was transcribed and the coding genes of alpha,beta and gamma subunit were amplified by polymerase chain reaction( PCR). Enzyme cutting sites in the both ends of alpha subunit were added,while enzyme cutting sites and connecting peptide in the both ends of beta and gamma subunits were added respectively. After enzyme digestion and connection,Giα1gene and Giβ1-γ2fusion gene were obtained. Giα1gene and Giβ1-γ2fusion gene were cloned into the p EYFP-N1 and p ECFP-C1 fluorescent protein reporter vectors. The eukaryotic expression vectors were transfected into Hep G2 liver cancer cells by lipofectamine method. The laser confocal microscope was used to observe and analyze their expressions in the liver cells. Results: The coding genes of alpha,beta and gamma subunit of Gi protein were amplified directly by PCR. The Giα1and Giβ1-γ2genes were obtained after enzyme digestion and connection,the eukaryotic expression vectors of p EYFP-N-Giα1and p ECFP-C-Giβ1-γ2were successfully constructed. Subsequently,the vectors were successfully transfected and expressed in the liver cancer cells. Conclusions: The eukaryotic expression vectors of Gi protein were successfully constructed and can express in the living cells.

关键词(KeyWords): Gi蛋白;脂质体;转染;荧光蛋白;真核表达载体
Gi protein;liposomes;transfection;fluorescent protein;eukaryotic expression vector

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(11162003,31260227,31170886);; 教育部科学技术研究重点项目(210196);; 贵州省优秀青年科技人才支持计划(2011-24);; 贵州省社会发展科技攻关项目[黔科合SY字(2011)3065];; 贵州省省长专项基金[黔省专合字(2009)-79];; 贵州省科学技术基金[黔科合J字2008-2274和(2013)2058];; 贵州省科技计划[黔科合LH字(2014)7092号];; 贵阳市科学技术项目(2010-筑科农合同字第1-社-12);; 贵州省留学回国人员科技活动基金(2013-8)

作者(Author): 胡祖权;何天军;贾义;宋萍萍;邱炜;赵远波;曾柱;
HU Zuquan;HE Tianjun;JIA Yi;SONG Pingping;QIU Wei;ZHAO Yuanbo;ZENG Zhu;Department of Biotechnology,College of Biology and Engineering,Guiyang Medical University;

Email:

DOI:

参考文献(References):

文章评论(Comment):

序号(No.) 时间(Time) 反馈人(User) 邮箱(Email) 标题(Title) 内容(Content)
反馈人(User) 邮箱地址(Email)
反馈标题(Title)
反馈内容(Content)
扩展功能
本文信息
服务与反馈
本文关键词相关文章
本文作者相关文章
中国知网
分享