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 |
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引用本文: | 杨 阳,黄 娜,高 岭,常素娥,郭 波,胡丽丽,宋土生,黄 辰.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[J].南方医科大学学报,2014,34(3):308-311. |
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作者姓名: | 杨 阳 黄 娜 高 岭 常素娥 郭 波 胡丽丽 宋土生 黄 辰 |
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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。
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Construction of pcDNA3-HERG-G572R expression vector and establishment of a cell linestably expressing HKE-HERG-G572R |
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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.
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