人hepcidin真核表达载体的构建及稳定转染细胞系的建立  

Construction of Human Hepcidin Eukaryotic Expression Vector and Establishment of Stably Transfected HepG2 Cell Line Expressing Human Hepcidin

作　　者：曾芳[1] 卢银平[2] 王凯平[1] 

机构地区：[1]华中科技大学同济医学院药学院药剂学系,武汉430030 [2]华中科技大学同济医学院附属协和医院病毒室,武汉430022

出　　处：《华中科技大学学报（医学版）》2010年第4期524-527,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：湖北省自然科学基金重点资助项目(No.2008CDA064)

摘　　要：目的构建人hepcidin全长结构基因真核表达载体,建立稳定高表达hepcidin的HepG2细胞系,以应用于拮抗hepcidin从而增强铁吸收的活性药物筛选。方法化学合成结合PCR拼接hepcidin全长结构基因,插入原核表达载体pMD18-T中,测序正确后再经HindⅢ与EcoRⅠ双酶切,定向插入真核表达载体pcDNA3.0内,酶切鉴定后,采用脂质体转染法将重组质粒转染人HepG2细胞,G418选择抗性细胞克隆,ELISA检测转染细胞hepcidin的蛋白表达。结果经酶切鉴定,成功构建hepcidin真核表达载体pcDNA3-hepcidin;重组质粒转染HepG2,经G418筛选3周获得抗性细胞克隆,其hepcidin蛋白表达量为普通HepG2细胞表达量的3倍。结论成功构建了稳定高表达hepcidin的HepG2细胞,该稳定转染细胞系的建立为进一步研究hepcidin降低肠道铁吸收的分子机制以及筛选拮抗hepcidin作用的活性药物奠定了实验基础。Objective To construct eukaryotic expression vector containing full length structural gene of human hepcidin and establish stably transfected HepG2 cell line with high expression of hepcidin. Methods The synthesized DNA encoding human hepcidin was inserted into vector pMD18-T after being identified by nucleotide sequencing. After being digested by HindⅢ and EcoR Ⅰ ,the purified hepcidin gene fragment was subeloned into the eukaryotic expression vector pcDNA3.0. After analysis by restrictive digestion reaction,the recombinant plasmid was transfected into HepG2 ceils with liposome. Resistant cell clones were selected with G418,and the expression of hepcidin in resistant clones was detected directly by ELISA. Results Recombi nant plasmid pcDNA3 hepcidin was successfully constructed as indicated by restriction endonuclease analysis. Resistant HepG2 cell clones were obtained by selection with G418 for 3 weeks. The expression yield of hepcidin was 3 times as much as that in common HepG2 cells. Conclusion The stably transfected HepG2 cell line highly expressing human hepcidin was successfully constructed. This lays the foundation for the further study on the molecular mechanism of hepcidin inhibiting iron export from enterocytes,and screening of active drugs that antagonize hepcidin.

关 键 词：人hepcidin 真核表达 全长结构基因 稳定转染 

分 类 号：R349.5[医药卫生—基础医学]