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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MKN45细胞中的表达
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【西药制药论文】【关键词】 大肠杆菌Construction of Eukaryotic Expression Vector Containing E.coli Purine Nucleoside Phosphorylase and Its Expression in MKN45 Cells
Abstract：Objective To clone E.coli PNP gene, construct its eukaryotic expression vector and obtain positive MKN45 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 pMD18T.After the sequencing was confirmed, the gene was subcloned to pcDNA3.1 to construct recombinant eukaryotic expression vector pcDNA3.1EPNP. The recombinant plasmid was transfected into MKN45 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. Resistan
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