Thrombotic Regulatory Protein Gene Control Arterial Injury, Thrombosis And Vascular Stenosis Experimental Research

Objective To construct eukaryotic expression plasmid containing human thrombomodulin gene for further study on hTM characteristic in anticoagulant, antiflammatory and neointima formation resistant. Methods The hTM gene expressive fragment was amplified by polymerase chain reaction (PCR) from human genomic DNA. Then, both pcDNA3.1(+)/neo vector and hTM fragment were digested by Hind III &EcoR I . Two digested fragments were ligated with T4DNA ligase. The product of reaction transferred E.coli line DH5a and cultured the bacteria on antibiotic plate. Recombinant plasmid was extracted from position clone and identified by redigesting, PCR and sequencing.Results The product of PCR from genome was a signal band about 1700bp. After identification of redigesting, PCR and sequencing, the recombinant plasmid was confirmed which contained the correct and full nucleotide sequence of hTM in pcDNA3.1(+)/neo. Conclusion The pcDNA3.1 /hTM plasmid was constructed.