Construction and expression of reconstructive plasmids with human thrombomodulin gene
- VernacularTitle:含人血栓调节蛋白基因重组质粒的构建和表达
- Author:
Yi DAI
;
Hui CHEN
;
Lin ZOU
;
Zhengrong QIAO
;
De SHI
- Publication Type:Journal Article
- Keywords:
Thrombomodulin;
Umbilical veins;
Endothelial cells;
Plasmids
- From:
Chinese Journal of Pathophysiology
2000;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
AIM: To provide experimental evidence for gene therapy of thrombophilia disease, we constructed the eukaryotic expression plasmid with human thrombomodulin (hTM) gene and observed the alteration of hTM expression on the surface of human umbilical vein endothelial cells (HUVECs) with and without the reconstructive plasmid. METHODS: The whole expressive fragment of hTM gene was amplified by PCR from human genome. Both hTM gene and pcDNA3.1(+)/neo empty vector was digested by HindⅢ and EcoRⅠ. Two digested fragments were ligated into pcDNA3.1/hTM with T_4DNA ligase. After identifying, the reconstructive plasmid transfected into HUVECs using lipofectin. The hTM antigen on the HUVECs was detected by immunohistochemistry. RESULTS: The hTM reconstructive plasmid was confirmed by double endonuclease redigesting and sequencing. About 10% HUVECs were transfected by pcDNA3.1/hTM plasmid with lipofectin and the high-level hTM was detected on the transfected cells. CONCLUSION: We constructed the pcDNA3.1/hTM plasmid successfully, and it could be expressed on the HUVECs. [
