南方医科大学学报
南方醫科大學學報
남방의과대학학보
JOURNAL OF SOUTHERN MEDICAL UNIVERSITY
2014年
3期
308-311
,共4页
杨阳%黄娜%高岭%常素娥%郭波%胡丽丽%宋土生%黄辰
楊暘%黃娜%高嶺%常素娥%郭波%鬍麗麗%宋土生%黃辰
양양%황나%고령%상소아%곽파%호려려%송토생%황신
HERG基因%HEK293%转染%突变体
HERG基因%HEK293%轉染%突變體
HERG기인%HEK293%전염%돌변체
HERG gene%HEK293 cell%transfection%mutation
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。
目的:構建HERG基因G572R突變體錶達載體併建立其穩定轉染HEK293的細胞繫。方法應用重疊延伸PCR構建HERG-G572R突變體錶達載體,經G418篩選穩定轉染細胞繫,通過Real-Time PCR,Western blot及測序鑒定穩定轉染細胞繫。結果經序列比對,證明HEK-HERG-G572R突變體錶達載體構建成功,Real-Time PCR,Western blot及測序證明穩定轉染細胞繫構建成功。結論該方法可成功構建的HKE-HERG-G572R細胞株,為藥物箇體化治療提供工具。
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.