人胰岛素样生长因子-1真核双顺反子表达载体pIRSES2-EGFP-hIGF-1的构建与鉴定(英文)  

Construction and Detection of Bicistronic Eukaryotic Vector pIRSES2-EGFP-hIGF-1

作　　者：续宗耀[1] 王英振[1] 张海宁[1] 吕成昱[1] 周峰[1] 

机构地区：[1]青岛大学医学院附属医院关节外科,山东青岛266003

出　　处：《现代生物医学进展》2013年第15期2837-2840,共4页Progress in Modern Biomedicine

基　　金：The National Natural Science Foundation of China(81171774;81272056)~~

摘　　要：目的:利用基因工程原理构建人胰岛素样生长因子1(hIGF-1)全长基因的真核表达载体pIRES2-EGFP-hIGF-1,并观察其在人胚肾293细胞中的表达。方法:应用PCR方法从人肝细胞文库中提取人hIGF-1全长cDNA序列,克隆入T-pMD18载体中,经测序证实序列正确后,利用XhoI/EcoRI进行双酶切并定向插入真核表达载体pIRES2-EGFP中,利用双酶切、电泳和PCR进行鉴定证实插入片段的正确性。应用高效转染试剂X-treme GENE HP DNA Transfection reagent介导真核表达载体pIRES2-EGFP-hIGF-1转染人胚肾细胞(HEK293),转染后利用倒置荧光显微镜与RT-PCR观察目的基因在靶细胞中的表达。结果:经酶切和测序证实重组质粒载体构建正确,转染后48小时可观察到绿色荧光的表达,RT-PCR结果证实转染pIRES2-EGFP-hIGF-1后可有hIGF-1基因靶细胞中表达。结论:成功构建真核表达载体pIRES2-EGFP-hIGF-1,目的基因与标记基因可同时在靶细胞中表达,为后续研究奠定基础。Objective： To construct the bicistronic eukaryotic expression recombinant plasmid pIRSES2-EGFP-hIGF-1 by gene engineering and to investigate its expression in human embryo kidney 293 cells（HEK293）.Methods： hIGF-1 cDNA was colned from liver cDNA library by polymerase chain reaction（PCR） and inserted into pMD18-T（TA Clone method）.The resultant plasmid was transfected into Escherichia coli TG-1 for amplification.After the DNA sequenced,XhoI and EcoRI were used to cut off the target gene and then inserted into the eukaryotic expression vector pIRSE2-EGFP by the two enzymes.The recombinant plasmid named pIRSES2-EGFP-hIGF-1 was verified by XhoI and EcoRI double-enzyme digestion analysis,and PCR.The plasmid was transfected into HEK293 cells by X-treme GENE HP DNA Transfection reagent in vitro.Then its expression was detection by using fluorescence microscopy and RT-PCR.Results： The hIGF-1 cDNA sequence and insert in recombinant eukaryotic expression plasmid pIRSES2-EGFP-hIGF-1 were both correct.48 hour later,the green fluorescence was observed to be emitted from HEK293 cells after transfection.The RT-PCR result demonstrated that hIGF-1 could express in HEK293 cells after transfected with pIRSES2-EGFP-hIGF-1.Conclusion： The vector pIRSES2-EGFP-hIGF-1 has been constrcuted successfully.The target gene and marker gene can be both expressed in the 293 cell lines,which lays the foundation for further research.

关 键 词：人胰岛素样生长因子1 基因 载体 转染 

分 类 号：Q75[生物学—分子生物学] Q78