CAJ | 학술논문

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。
목적：구건HERG기인G572R돌변체표체재체병건립기은정전염HEK293적세포계。방법응용중첩연신PCR구건HERG-G572R돌변체표체재체，경G418사선은정전염세포계，통과Real-Time PCR，Western blot급측서감정은정전염세포계。결과경서렬비대，증명HEK-HERG-G572R돌변체표체재체구건성공，Real-Time PCR，Western blot급측서증명은정전염세포계구건성공。결론해방법가성공구건적HKE-HERG-G572R세포주，위약물개체화치료제공공구。
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.