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Cloning of Coding Fragment of Human Thrombomodulin Gene and Expression in Eukaryotic Cells

【Abstract】Objective To provide experimental evidence for gene therapy of thrombophilia diseases by constructing eukaryotic expression plasmid of human thrombomodulin(hTM) gene and transfecting the plasmid into COS7 cell and human umbilical vein endothelial cells(HUVECs). Methods The coding fragment of hTM gene was amplified by PCR. Both hTM gene and pcDNA3.1(+)/neo empty vector were digested with HindⅢ and EcoRⅠ. Two digested fragments were combined into pcDNA3.1/hTM with T4DNA ligase. After identification, the pcDNA3.1/hTM was transfected into COS7 cell and HUVECs using cation liposome. The expression of hTM mRNA and protein on the COS7 cell and HUVECs was detected by RTPCR and immunohistochemistry respectively. Results The hTM recombinant plasmid was confirmed by double endonuclease digesting and sequencing. It was transfected into COS7 cell and HUVECs successfully with liposome.Conclusion The pcDNA3.1/hTM plasmid can be successfully constructed and highlevel hTM can be expressed in eukaryotic cells. All of this provides us experimental evidence for gene therapy and further study of TM anticoagulant mechanism.