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

Objective To clone E.coli PNP gene, construct its eukaryotic expression vector and obtain positive MKN-45 cell clones expressing E.coli PNP gene stably. 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 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 plasmid was transfected into MKN-45 cells 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.coli PNP gene was successfully constructed. The positive MKN-45 cell clones expressing E.coli 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.