南方医科大学学报

南方医科大学学报 ›› 2014, Vol. 34 ›› Issue (03): 308-.

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HERG基因G572R突变体表达载体的构建及其稳定转染细胞系的建立

杨阳，黄娜，高岭，常素娥，郭波，胡丽丽，宋土生，黄辰   

  • 出版日期:2014-03-20 发布日期:2014-03-20

Construction of pcDNA3-HERG-G572R expression vector and establishment of a cell line
stably expressing HKE-HERG-G572R

  • Online:2014-03-20 Published:2014-03-20

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摘要： 目的构建HERG基因G572R突变体表达载体并建立其稳定转染HEK293 的细胞系。方法应用重叠延伸PCR构建
HERG-G572R突变体表达载体，经G418筛选稳定转染细胞系，通过Real-Time PCR，Western blot及测序鉴定稳定转染细胞系。
结果经序列比对，证明HEK-HERG-G572R突变体表达载体构建成功，Real-Time PCR，Western blot 及测序证明稳定转染细胞
系构建成功。结论该方法可成功构建的HKE-HERG-G572R细胞株，为药物个体化治疗提供工具。

Abstract: Objective To construct the pcDNA3-HERG-G572R expression vector and establish a cell line stably expressing
HKE-HERG-G572R. Methods HERG-G572R mutant fragment was constructed by over-lap extension PCR and validated by
DNA sequencing. The HKE-HERG-G572R expression vector was constructed and transfected into HEK293 cells to obtain a cell
line stably expressing HKE-HERG-G572R. Results The pcDNA3-HERG-G572R expression vector was successfully constructed
and the cell line stably expressing HKE-HERG-G572R was established. Real-time PCR and Western blotting revealed a 632-fold
HKE-HERG-G572R overexpression in the transfected HEK293 cells as compared with that in control HEK293 cells transfected
with pcDNA3 (P<0.01)．Conclusion The protocol can be used to construct the cell line stably expressing HKE-HERG-G572R to
provide a cell model for studying individualized therapy.