大肠杆菌胞嘧啶脱氨酶基因的克隆及其真核表达载体的构建

目的 克隆大肠杆菌胞嘧啶脱氨酶(CD)基因，构建真核表达载体，本研究拟探索该基因在肿瘤基因治疗中的应用基础。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，并与pcDNA3．1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pcDNA3．1-CD，并用限制性内切酶、PCR和DNA测序进行鉴定。结果克隆了大肠杆菌CD基因，并构建了真核表达载体，经限制性内切酶酶切、PCR扩增和DNA测序证实了其正确性。结论 pcDNA3．1-CD真核表达载体构建成功。

The cloning of cytosine deaminase gene of Escherichia coli and the construction of eukaryotic expression vector

Objective To clone cytosine deaminase(CD) gene of Escherichia coli and to construct its eukaryotic expression vector Methods A pair of primers were designed and synthesized based on CD gene sequence in GenBank And CD gene was cloned from Escherichia coli genome by PCR amplification The PCR products were cloned into a eukaryotic plasmid pcDNA3 1 to construct the recombination expression vector which was controlled by CMV promoter The recombinant plasmid was analyzed and identified by PCR, restriction digest and sequencing Results The CD gene was cloned and eukarytoic expression vector (pcDNA3 1 CD) was constructed It was demonstrated that CD gene was properly inserted into the vector and the sequence was confirmed with restriction digest, PCR amplification and sequencing Conclusion The eukaryotic expression vector with CD gene (pcDNA3 1 CD) was successfully constructed