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

作　　者：徐婧[1] 侯赋园 王德彬[1] 李竣[1] 胡鑫[1] 杨光忠[1] Xu Jing;Hou Fuyuan;Wang Debin;Li Jun;Hu Xin;Yang Guangzhong(School of Pharmaceutical Sciences,South-Central University for Nationalities,Wuhan 430074,China)

机构地区：[1]中南民族大学药学院,武汉430074

出　　处：《中南民族大学学报（自然科学版）》2018年第3期28-31,共4页Journal of South-Central University for Nationalities：Natural Science Edition

基　　金：国家自然科学基金资助项目(31302172);中央高校基本科研业务费专项资金资助项目(CZP17029)

摘　　要：目的:构建pEGFP-C1-MCH真核表达载体,并将其转染入HEK293细胞中,筛选阳性细胞克隆,为研究MCH基因在能量代谢中的功能及机制提供细胞模型.方法:提取脑组织总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.再将该基因全长cDNA克隆至质粒pEGFP-C1,经菌落PCR筛选及双酶切和DNA测序鉴定,成功构建了含有目的基因MCH的重组质粒pEGFP-C1-MCH.并利用脂质体2000介导其转染HEK293细胞,用荧光显微镜和RT-PCR检测EGFP和MCH在细胞中的表达.结果:克隆的pEGFP-C1-MCH质粒序列中的MCH与Gen Bank相符;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,MCH基因稳定表达.结论: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实验模型.Objective:To study the function and mechanism of MCH gene in energy metabolization,construct the eukaryotic expression vector pEGFP-C1-MCH and transfect it into HEK293 cell lines,then screen out the positive cell clone.Methods: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 recombinant plasmid of pEGFP-C1-MCH containing MCH gene was successfully constructed and was transfected into HEK293 cells using Lipofectamine 2000.The results of transfection were analyzed using fluorescence microscopy and RT-PCR.Results:The sequence of the cloned plasmid(pEGFP-C1-MCH)was in accordance with the GenBank.After 72 h transfection,the cells transfected successfully expressed green fluorescence,and MCH gene was stably expressed.Conclusion: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.

关 键 词：黑色素聚焦激素 绿色荧光蛋白 PEGFP-C1 转染 HEK293 

分 类 号：Q786[生物学—分子生物学]