摘要
目的构建HERG基因的真核表达载体,并在人胚胎肾细胞(HEK293)中进行表达。方法先将pGH19-HERG通过限制性酶SacI和EcoRI酶切得到HERGcDNA,将pIRES2-EGFP用SacI和EcoRI进行双酶切,把HERGcDNA定向克隆到pIRES2-EGFP中,即构建了HERG基因的真核表达载体pIRES2-EGFP-HERG。然后利用电穿孔法将pIRES2-EGFP-HERG转染HEK293细胞。结果在卵母细胞异源表达载体pGH19-HERG基础上,获得了真核表达载体pIRES2-EGFP-HERG,并在HEK293细胞中成功进行了表达。结论在HERG基因卵母细胞异源表达载体的基础上,构建了HERG基因的真核表达载体pIRES2-EGFP-HERG,利用电穿孔法将其转染至HEK293细胞中并成功地进行了表达,为下一步进行膜片钳的研究奠定基础。
[Objective] To construct the eukaryotic expression vector of the HERG gene and express it in HEK293 cells. [Methods] HERG cDNA was obtained from pGH19-HERG by restriction endonucleases Sac I and EcoR I digestion and was inserted into the same restriction site of pIRES2-EGFP in the same way, thus pIRES2- EGFP-HERG was constructed. The recombinant plasmids pIRES2-EGFP-HERG were identified by sequencing, pIRES2-EGFP-HERG was transfected into HEK293 cells by electroporation. [Results] The eukaryotic expression vector pIRES2-EGFP-HERG was constructed correctly and expressed in HEK293 cells. [Conclusions] The recombined pIRES2-EGFP-HERG eukaryotic expression vector can be expressed in HEK293 cells. These results make a foundation for the further functional study of HERG gene.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2008年第3期332-334,共3页
China Journal of Modern Medicine
