大肠杆菌嘌呤核苷磷酸化酶基因的克隆与真核表达载体的构建

根据Ｇｅｎｂａｎｋ中大肠杆菌嘌呤核苷磷酸化酶（ＰＮＰ）基因的核苷酸序列，设计并合成了一对引物，以大肠杆菌基因组ＤＮＡ为模板，进行ＰＣＲ扩增，并将扩增产物定向连接到克隆、测序及真核表达载体ＰＣＤＮＡ３中，进行酶切鉴定、测序及序列分析。结果表明ＰＣＲ扩增出７４１ｂｐ大小的片段，通过酶切和序列分析证明含完整的ＰＮＰ基因序列且基因插入方向正确，此序列与文献报道的ＰＮＰ基因的同源性为９９．７％。说明克隆的ＰＮＰ基因与文献报道的基本一致，ｐｃＤＮＡ３－ＰＮＰ的构建成功为今后用其进行基因转染来研究ＰＮＰ／Ｍｅｐ－ｄＲ自杀基因系统在肿瘤基因治疗中的应用打下了基础。

Cloning and construction of E.coli purine nucleoside phosphorylase gene into an eukaryotic expressing vector

Objective: To amplify the E. coli PNP gene and clone into an eukaryotic expressing plasmid. A high effective expressing vector containing E. coli PNP gene would be constructed. Methods: PCR amplification was performed using primers based on E. coli PNP gene sequence from Genbank, E. coli genomic DNA as template. PCR product was directionally ligated into a cloning, sequencing, and high effective eukaryotic expressing plasmid pcDNA3. Sequencing of PNP gene from this vector was performed and analyzed. Results: A 741bp DNA fragment was amplified. Recombinant of pcDNA3-PNP was constructed. Sequence and restriction endonuclease analysis demonstrated that the cloned fragment contained complete E. coli PNP gene and indicated a 99. 7% identity between the cloned PNP gene and that from Genbank. Conclusion: The cloned PNP gene was nearly identical with that from Genbank. The success of pcDNA3-PNP recombi- nant has laid a solid ground for the further study of cancer gene therapy with PNP/Mep-dR sui- cide gene system.