Construction of a HERG mutant L539fs/47-*558W pEGFP vector and the expression of the fusion protein in HEK293 cells  

作　　者：ZHANG Junbo Lü Ying ZHANG Aifeng SUN Chaofeng HAN Wenqi LI Guoliang GAO Jie HUO Jianhua PAN Junqiang ZHOU Xin NIU Xiaolin 

机构地区：[1]Department of Cardiovascular Medicine, First Affiliated Hospital, Institute of Cardiovascular Channelopathy, Ministry of Education Key Laboratory of Environment and Genes Related to Diseases, Xi'an Jiaotong University, Shaanxi Key Laboratory of Molecular Cardiology [2]Department of Cardiovascular Medicine, Second Affiliated Hospital, Xi'an Jiaotong University

出　　处：《Journal of Medical Colleges of PLA(China)》2013年第4期193-205,共13页中国人民解放军军医大学学报（英文版）

基　　金：Supported by the National Natural Science Foundation of China(No.30800473)

摘　　要：Objective： To construct a human ether-a-go-go-related gene （HERG） nonsense mutant L539fs/47-558W into the autonomously fluorescent, eukaryotic expression vector pEGFP-C2, and to verify expression of the reconstruct in human embryonic kidney-293 （HEK293） cells. Methods： The mutational fragment was subcloned into pEGFP-C2-HERG by double digestion of Sbf I, Eco91 1 and rejoining of T4 ligase. After verification, the recombinant pEGFP-C2-L539fs/47-558W and pEGFP-C2-HERG were respectively transfected into HEK293 cells for 48 h by the Lipofect method to observe the expression location of the fusion protein by laser confocal imaging scanning in vivo. pcDNA3 -L539fs/47-＊558W and pcDNA3-HERG were transfected to observe the expression location of the HERG protein by immunofluoresceoce. The mutant protein size was determined by Western blotting. Results： The about 1 kb-sized mutation region cDNA fragment from pcDNA3-L539fs/47-＊558Wand the about 7.2 kb-sized target vector fragment from pcDNA3-HERG were ligated after purification and gel recovery pEGFP-C2-L539fs/47＊-558W, approximately 8.2 kb, was demonstrated successfully been constructed under agarose gel electrophoresis and further sequencing. Laser confocal imaging showed that pEGFP-C2-HERG was mainly expressed in the membrane, whereas truncated mutant-type HERG in the pEGFP-C2 vector was partially located in the cytoplasm, the others were transported to the cell membrane in living HEK293 cells. The same as the immunofluoresceoce results after transfection of pcDNA3-HERG and pcDNA3-L539fs/47-558W. Wild-type HERG-GFP fusion protein expressed 160 and 180 kDa bands. The mutant and mutant-GFP fusion proteins were 70 and 100 kDa, respectively. Conclusion： pEGFP-C2-L539fs/47-＊558W was successfully constructed by double digestion method GFP had no effect on its protein expression and trafficking in HEK293 cells, which laid a foundation for the further study on L539fs/47-＊558WObjective:To construct a human ether-a-go-go-related gene(HERG)nonsense mutant L539fs/47-*558W into the autonomously fluorescent,eukaryotic expression vector pEGFP-C2,and to verify expression of the reconstruct in human embryonic kidney-293(HEK293)cells.Methods:The mutational fragment was subcloned into pEGFP-C2-HERG by double digestion of SbfⅠ,Eco91Ⅰand rejoining of T4 ligase.After verification,the recombinant pEGFP-C2-L539fs/47-*558W and pEGFP-C2-HERG were respectively transfected into HEK293 cells for 48 h by the Lipofect method to observe the expression location of the fusion protein by laser confocal imaging scanning in vivo.pcDNA3-L539fs/47-*558W and pcDNA3-HERG were transfected to observe the expression location of the HERG protein by immunofluoresceoce.The mutant protein size was determined by Western blotting.Results:The about 1 kb-sized mutation region cDNA fragment from pcDNA3-L539fs/47-*558W and the about 7.2 kb-sized target vector fragment from pcDNA3-HERG were ligated after purification and gel recovery.pEGFP-C2-L539fs/47*-558W,approximately 8.2 kb,was demonstrated successfully been constructed under agarose gel electrophoresis and further sequencing.Laser confocal imaging showed that pEGFP-C2-HERG was mainly expressed in the membrane,whereas truncated mutant-type HERG in the pEGFP-C2 vector was partially located in the cytoplasm,the others were transported to the cell membrane in living HEK293 cells.The same as the immunofluoresceoce results after transfection of pcDNA3-HERG and pcDNA3-L539fs/47-558W.Wild-type HERG-GFP fusion protein expressed 160 and 180 kDa bands.The mutant and mutant-GFP fusion proteins were 70 and 100 kDa,respectively.Conclusion:pEGFP-C2-L539fs/47-*558W was successfully constructed by double digestion method GFP had no effect on its protein expression and trafficking in HEK293 cells,which laid a foundation for the further study on L539fs/47-*558W.

关 键 词：Human ether-a-go-go-related gene MUTATION Eukaryotic expression vector PEGFP-C2 

分 类 号：R341[医药卫生—基础医学]