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目的克隆多药耐药基因(MDR1)启动子,构建并鉴定MDR-1启动子靶向的CD、TK双自杀基因表达载体。方法提取耐药胶质瘤细胞株C6/ADR的基因组DNA,通过PCR方法选择性扩增多药耐药MDR1启动子片段,连接到T载体上,酶切后定向克隆入pcDNA3.TK载体上的CMV启动子位置,然后从pcDNA3.CD.TK上酶切下CD片段并插入上述载体形成pcDNA3.MDR1P.CDTK,通过电泳及DNA测序对其进行鉴定。结果通过PCR方法扩增出了MDR1启动子片段,并连接到T载体上,然后成功克隆入pcDNA3.TK中,形成pcDNA3.MDR1P.TK,将pcDNA3.CD.TK上的CD基因切下后插入pcDNA3.MDR1P.TK,电泳及DNA测序证实连接准确。结论成功构建了含正确MDR1启动子靶向的双自杀基因表达载体pcDNA3.MDR1P.CDTK,为今后靶向治疗耐药肿瘤构建载体提供了实验基础。
Objective To clone the multidrug resistance gene (MDR1) promoter and construct a dual CDK suicide gene expression vector targeting MDR-1 promoter. Methods The genomic DNA of drug-resistant glioma cell line C6 / ADR was extracted. The multidrug-resistant MDR1 promoter fragment was amplified by PCR and ligated into T vector. After digestion, the vector was cloned into the pcDNA3.TK vector CMV promoter. The CD fragment was then digested from pcDNA3.CD.TK and inserted into the above vector to form pcDNA3.MDR1P.CDTK, which was identified by electrophoresis and DNA sequencing. Results The MDR1 promoter fragment was amplified by PCR and ligated into T vector and then cloned into pcDNA3.TK successfully to form pcDNA3.MDR1P.TK. The CD gene on pcDNA3.CD.TK was cut and inserted pcDNA3.MDR1P.TK, electrophoresis and DNA sequencing confirmed that the connection is accurate. Conclusion The double suicide gene expression vector pcDNA3.MDR1P.CDTK containing the correct MDR1 promoter was successfully constructed, which provided the experimental basis for the future targeting of drug-resistant tumor-building vector.