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目的 克隆大肠杆菌胞嘧啶脱氨酶 (CD)基因 ,构建真核载体并转染食管癌细胞系 EC1 0 9,观察大肠杆菌胞嘧啶脱氨酶 / 5 -氟胞嘧啶 (CD/ 5 - FC)自杀基因系统杀伤肿瘤细胞以及对放射治疗的增敏效应。 方法 根据 Genbank数据库提供的 CD基因核苷酸序列 ,设计并合成一对引物 ,采用PCR方法 ,从大肠杆菌基因组 DNA中扩增出 CD基因 ,与pc DNA3.1定向连接 ,构建受控于人巨细胞病毒启动子的重组真核载体 pc DNA3.1 - CD,并用限制性内切酶、PCR和DNA测序进行鉴定 ,用脂质体转染法转染食管癌细胞系EC1 0 9细胞 ,用 G4 1 8筛选后获得阳性克隆株 EC1 0 9- CD,经PCR进行鉴定 ,CD/ 5 - FC体系对 EC1 0 9细胞的杀伤效应和杀伤时的旁观者效应以及对放射治疗的增敏效应 ,MTT法检测细胞存活比率。 结果 克隆大肠杆菌 CD基因 ,构建真核表达载体 ,经限制性内切酶酶切、PCR扩增和 DNA测序证实其正确性 ,CD基因转染 EC1 0 9细胞后 ,经 G4 1 8筛选后获得阳性克隆株 EC1 0 9- CD,经 RTP- CR分析表明 CD基因已转入 EC1 0 9细胞并开始转录。体外实验表明 ,CD/ 5 - FC体系的旁观者效应及对放射治疗增敏效果明显。 结论 pc DNA3.1 - CD真核表达载体构建及转染 EC1 0 9细胞成功 ,CD/ 5 - FC体系的旁观者效应和放射治疗增敏效果明
Objective To clone the CD gene of Escherichia coli and construct eukaryotic vector and transfect it into esophageal cancer cell line EC1 0 9 to observe the effects of E. coli CDase / 5 - fluorocytosine (CD / 5 - FC) Suicide gene systems kill tumor cells and sensitize to radiation. Methods According to the nucleotide sequence of CD gene provided by Genbank database, a pair of primers was designed and synthesized. The CD gene was amplified from the genomic DNA of E. coli by PCR and ligated with pc DNA3.1 to construct a The recombinant eukaryotic vector pcDNA3.1 - CD was constructed and identified by restriction endonuclease, PCR and DNA sequencing. The transfected esophageal cancer cell line EC1 0 9 was transfected with G4 The positive clone EC1 0 9-CD was screened and identified by PCR. The killing effect of CD / 5 - FC system on EC1 0 9 cells and the bystander effect during killing and the sensitization effect to radiotherapy were observed. MTT Method to test cell survival rate. Results The CD gene of E. coli was cloned and the eukaryotic expression vector was constructed. The correctness of restriction endonuclease digestion, PCR amplification and DNA sequencing were confirmed. CD gene was transfected into EC1 0 9 cells and screened by G418 The positive clone EC1 0 9-CD, analyzed by RTP-CR, showed that the CD gene had been transferred into EC1 0 9 cells and started to be transcribed. In vitro experiments show that the bystander effect of CD / 5 - FC system and the sensitization effect to radiation therapy are obvious. Conclusion The construction of pcDNA3.1 - CD eukaryotic expression vector and the successful transfection of EC1 0 9 cells, the bystander effect of CD / 5 - FC system and the radiosensitization effect