摘要
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 pEYFP-N-Giα1 and pECFP-C-Giβ-γ2 ; detecting the expression of vectors in the eukaryotic cells.Methods：HepG2 liver cancer cells were cultured and the total RNA was isolated.Then cDNA 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,Giα1 gene and Giβ1-γ2 fusion gene were obtained.Giα1 gene and Giβ1-γ2 fusion gene were cloned into the pEYFP-N1 and pECFP-C1 fluorescent protein reporter vectors.The eukaryotic expression vectors were transfected into HepG2 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 Giα1 and Giβ1-γ2 genes were obtained after enzyme digestion and connection,the eukaryotic expression vectors of pEYFP-N-Giα1 and pECFP-C-Giβ1-γ2 were 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.
出处
《贵阳医学院学报》
CAS
2014年第6期794-799,共6页
Journal of Guiyang Medical College
基金
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)
关键词
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
