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.pdfVIP

·308 · doi 10.3969/j.issn. 1673-4254.2014.03.04 J South Med Univ, 2014, 34(3): 308-311 基础研究 HERGHERG基因基因GG572572RR 突变体表达载体的构建及其稳定转染细胞系突变体表达载体的构建及其稳定转染细胞系 的建的建立立 1 2 1 1 1 1 1 1，3 杨 阳 ，黄 娜 ，高 岭 ，常素娥 ，郭 波 ，胡丽丽 ，宋土生 ，黄 辰 1 2 西安交通大学医学院遗传学与分子生物学系，陕西 西安 710061；西安交通大学第二附属医院科研实验中 3 心，陕西 西安 710004； 西安交通大学医学院心血管研究中心，陕西 西安 710061 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。 关键词：HERG基因；HEK293；转染；突变体 Construction of pcDNA3- HERG- G572R expression vector and establishment of a cell line stably expressing HKE-HERG-G572R 1 2 1 1 1 1 1 1, 3 YANG Yang , HUANG Na , GAO Ling , CHANG Sue , GUO Bo , HU Lili , SONG Tusheng , HUANG Chen 1Department of Genetics and Molecular Biology, 3Cardiovascular Research Center, Xian Jiaotong University College of Medicine, Xian 710061, China; 2Second Hospital of Xian Jiaotong University, Department of Genetics and Molecular Biology, Medical School, Xian Jiaotong University, 710004, China 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