大肠杆菌嘌呤核苷磷酸化酶基因真核表达载体的构建及其在MKN-45细胞中的表达

Construction of Eukaryotic Expression Vector Containing E.coli Purine Nucleoside Phosphorylase and Its Expression in MKN-45 Cells

目的克隆大肠杆菌嘌呤核苷磷酸化酶(EPNP)基因并构建真核表达载体pcDNA3.1-EPNP,将重组质粒转染MKN-45细胞获得高表达大肠杆菌PNP基因的MKN-45细胞克隆。方法根据Gen-bank中大肠杆菌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,并将扩增产物定向插入pMD-18T克隆载体中进行序列测定,测序正确后将其亚克隆至表达载体pcDNA3.1,利用脂质体将重组质粒转染MKN-45细胞。结果PCR扩增出716bp大小的片段,测序结果与已报道的EPNP基因序列一致;亚克隆经酶切鉴定正确;RT-PCR证明经G418筛选得到的转基因MKN-45细胞克隆中存在大量EPNPmRNA,前体药MePdR对转染EPNP的MKN-45细胞有较强的细胞毒作用。结论成功构建了大肠杆菌PNP基因的真核表达载体,获得了稳定表达大肠杆菌PNP基因的MKN-45细胞克隆,为PNP/MepdR自杀基因系统在胃癌基因治疗中的应用奠定了良好的基础。

Objective To clone E. coil PNP gene, construct its eukaryotic expression vector and obtain positive MKN-45 cell clones expressing E. coil PNP gene stably. Methods PCR amplification was performed using primers based on E. coil PNP gene sequence from Genbank, E. coil genomic DNA as template. PCR product was inserted into pMD-18T. After the sequencing was confirmed, the gene was subcloned to pcDNA3. 1 to construct recombinant eukaryotic expression vector pcDNA3. 1-EPNP. The recombinant piasmid was transfected into MKN-45 ceils by lipofectamin method. Results PCR yielded a fragment of 716bp and EPNP was verified by sequence analysis. Enzyme digestion analysis showed that the target gene was sub cloned into recombinant vector. Resistant clones MKN-45 expressed high amounts of EPNP mRNA and showed a strong sensitivity to MePdR. Conclusion The eukaryotic expression plasmid containing E. coil PNP gene was successfully constructed. The positive MKN-45 cell clones expressing E. coil PNP gene stably were obtained, which has laid a solid ground for further study of gastric cancer gene therapy with PNP/MepdR suicide gene system.