Eukaryotic expression vector of heme oxygenase-1 and its expression in endothelial cell

• Published in: Hepatobiliary & pancreatic diseases international Vol. 3; no. 3; pp. 360 - 364
• Main Authors: Du, Dun-Feng, Chang, Sheng, Chen, Bi-Cheng, Chen, Zhong-Hua
• Format: Journal Article
• Language: English
• Published: Singapore 01.08.2004
• Subjects: Animals; Blotting, Western; cell; clone; Cloning, Molecular; DNA, Complementary - genetics; endothelial; Endothelium, Vascular - cytology; Endothelium, Vascular - physiology; expression; Fluorescent Antibody Technique, Indirect; gene; Gene Expression; Genetic Vectors - genetics; heme; Heme Oxygenase (Decyclizing) - genetics; Heme Oxygenase-1; oxygenase-1; Plasmids - genetics; Rats; Rats, Wistar; Recombinant Proteins - genetics; Spleen - physiology; Transfection
• ISSN: 1499-3872

Heme oxygenase-1 (HO-1)-mediated cytoprotection may inhibit or postpone the formation of graft arteriosclerosis. The aim of this study was to construct eukaryotic expression vector of HO-1 gene and express recombinant HO-1 in human endothelial cells of the umbilical vein to provide an important clue for chronic rejection study. HO-1 gene was amplified from rat spleen with reverse transcription-polymerase chain reaction, and then cloned into eukaryotic expression vector pcDNA3 by means of recombinant gene technology. The recombinant plasmid pcDNA3-HO-1 was transfected into endothelial cells, and recombinant HO-1 was expressed in the endothelial cells under G418 selection. The expressed products were detected using indirect fluorescent staining and Western blot analysis. Restriction analysis indicated that the HO-1 gene was successfully inserted into the expression vector, and DNA sequencing verified that the reading frame of the recombinant vector was correct. Recombinant HO-1 was successfully expressed in endothelial cells and its molecular weight was about 32 kD. The successful construction of eukaryotic expression vector containing the HO-1 gene and the effective expression of recombinant HO-1 in endothelial cells has laid a foundation for further study on its biological functions.