人EGFR显性负性突变体真核表达载体的构建、蛋白表达及亚细胞结构定位
Construction of the eukaryotic vector carrying human dominant negative epidermal growth factor receptor,its expression and sub-cellular localization in COS-7 cells
廖刚 1王子卫 1赵林 1张能 1汤为学2
作者信息
- 1. 重庆医科大学附属第一医院胃肠外科,重庆,400016
- 2. 重庆医科大学基础医学院病理生理学教研室,重庆,400016
- 折叠
摘要
目的:构建人EGFR显性负性突变体真核表达载体pEGFPN1-DNEGFR,转染COS-7细胞,检测DNEGFR-EGFP的表达并进行亚细胞结构定位.方法:将RT-PCR(reverse transcription-polymerase chain reaction)方法扩增得到编码EGFR信号肽段、胞外区和跨膜区的cDNA,定向克隆至空载体pEGFP-N1中,构建真核表达载体pEGFPN1-DNEGFR.经PCR扩增鉴定、双酶切鉴定、核苷酸序列测定以及生物信息学分析证明pEGFPN1-DNEGFR构建成功后,脂质体法转染体外培养的COS-7细胞,Western Blot检测DNEGFR-EGFP蛋白的表达,应用光谱激光扫描共聚焦显微镜对DNEGFR-EGFP亚细胞结构定位检测.结果:PCR扩增鉴定、双酶切鉴定、核苷酸序列测定以及生物信息学分析证实pEGFPN1-DNEGFR构建成功,并且Western Blot检测DNEGFR-EGFP蛋白的表达,光谱激光扫描共聚焦显微镜观察显示DNEGFR-EGFP蛋白主要定位于细胞膜.结论:成功构建人EGFR显性负性突变体真核表达载体,并在COS-7细胞胞膜上表达,为靶向EGFR显性负性策略在肿瘤基因治疗中的进一步研究打下基础.
Abstract
AIM:To construct the eukaryotic expression vector (pEGFPN1-DNEGFR)carrying human dominant negative epidermal growth factor receptor(DNEGFR),detect the expression and the sub-cellular localization of dominant negative epidermal growth factor receptor-enhanced green fluorescence protein(DNEGFREGFP)in COS-7 cells transfected with pEGFPN1-DNEGFR.METHODS:The cDNA coding signal peptide,extracellular ligand-binding domain and membrane-spanning region of epidermal growth factor receptor(EGFR)precursor was obtained by reverse transcription-polymerase chain reaction(RT-PCR),then directionally cloned into the empty vector(pEGFP-N1)to construct pEGFPN1-DNEGFR.After confirmed by PCR amplification assay,double enzyme digestion,DNA sequencing and bioinformatics analysis of nucleotide sequence,pEGFPN1-DNEGFR was transfected into COS-7 cells,mediated by Lipofectamine 2000.The expression of and the sub-cellular localization of DNEGFREGFP in COS-7 cells were detected by Western Blot and Laser Scanning Spectral Confocal Microscope respectively.RESULTS:pEGFPN1-DNEGFR was constructed successfully,which was confirmed by PCR amplification assay,double enzyme digestion,DNA sequencing and bioinformatics analysis of nucleotide sequence.The expression of DNEGFR-EGFP was verified by Western Blot,and its predominant localization on the cell membrane of COS-7 cells was identified by Laser Scanning Spectral Confocal Microscope.CONCLUSION:The suecessful constrnction of pEGFPN1-DNEGFR,the expression and the sub-cellular localization of DNEGFR-EGFP in COS-7 cells,laid a solid foundation for further research on EGFR-targeted dominant negative strategy in cancer gene therapy.
关键词
表皮生长因子受体/显性负性突变体/单核苷酸多态性/定向克隆/亚细胞结构定位Key words
epidermal growth factor receptor/dominant negative mutant/single nucleotide polymorphism/directional cloning/sub-cellular localization引用本文复制引用
出版年
2009
NETL
NSTL
维普
万方数据