pEGFP-C1-MCH真核表达载体的构建及其在HEK293 细胞系中的表达
Construction of pEGFP-C1-MCH Eukaryotic Expression Plasmid and Its Expression in HEK293 Cell Line
投稿时间:2016-12-16  最后修改时间:2017-03-22
DOI:
中文关键词: MCH  绿色荧光蛋白  pEGFP-C1  转染  HEK293
英文关键词: MCH, green  fluorescent protein, pEGFP-C1, transfection, HEK293
基金项目:国家自然科学基金资助项目(31302172);湖北省民族药物现代化工程技术研究中心资助项目(2015ZY004)
作者单位E-mail
杨光忠 中南民族大学药学院 xuj@mail.scuec.edu.cn 
王德彬 中南民族大学药学院  
李竣 中南民族大学药学院  
胡鑫 中南民族大学药学院  
徐婧 中南民族大学药学院  
摘要点击次数: 45
全文下载次数: 16
中文摘要:
      构建pEGFP-C1-MCH真核表达载体,并将其转染入HEK293细胞中,筛选出阳性细胞克隆,为研究MCH基因在能量代谢中的功能及机制提供细胞模型。提取脑组织中的总RNA,反转录为cDNA,参照Genbank中提供的序列设计引物扩增MCH基因全长。然后将该基因全长cDNA克隆至真核表达载体pEGFP-C1,经菌落PCR筛选及双酶切和测序鉴定,构建含有目的基因MCH的重组质粒pEGFP-C1-MCH。利用脂质体介导pEGFP-C1-MCH质粒转染HEK293细胞,荧光显微镜和RT-PCR检测EGFP和MCH在细胞中的表达。双酶切和测序结果表明,克隆的MCH质粒序列和方向与预期相符,质粒载体构建成功。细胞转染72 h后,转染成功的细胞在荧光显微镜下表达较强的绿色荧光,RT-PCR结果显示细胞转染后MCH基因稳定表达。pEGFP-C1-MCH真核表达载体的构建及其在HEK293细胞中的稳定表达,为研究MCH基因在能量代谢中的功能及作用机制提供了有用的细胞模型。
英文摘要:
      To study the function and mechanism of MCH gene in energy metabolization, the eukaryotic expression vector pEGFP-C1-MCH was constructed and transfected into HEK293 cell lines, then the positive cell clone was screened out. Total RNA was extracted from the brain tissue and then reverse-transcribed to cDNA. The full-length cDNA of MCH was amplified with primers designed according to the sequence published in Genbank and inserted into the plasmid pEGFP-C1. The plasmid was screened by colony PCR and verified by restriction enzyme analysis and DNA sequencing. The eukaryotic expression vector pEGFP-C1-MCH was successfully constructed. The recombinant plasmid pEGFP-C1-MCH was transfected into HEK293 cells using Lipofectamine 2000. The results of transfection were analyzed using fluorescence microscopy and RT-PCR. Double digestion and DNA sequencing indicated that the sequence of the constructed plasmid (pEGFP-C1-MCH) was in accordance with the GenBank. After 72 h transfection, the cells transfected successfully expressed green fluorescent protein and MCH gene. The construction of pEGFP-C1-MCH recombinant plasmid and its stable expression in HEK293 cells provide an experimental model to explore the function and mechanism of MCH gene in energy metabolization.
View Fulltext   查看/发表评论  下载PDF阅读器
关闭