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| Eukaryotic expression vector of heme oxygenase-1 and its expression in endothelial cell |
| Dun-Feng Du, Sheng Chang, Bi-Cheng Chen and Zhong-Hua Chen |
Wuhan, China
Author Affiliations: Institute of Organ Transplantation, Affiliated Tongji Hospital, Tongji Medical College, Huazhong Science and Technology University, Wuhan 430030, China (Du DF, Chang S, Chen BC and Chen ZH)
Corresponding Author: Dun-Feng Du, MD, Institute of Organ Transplantation, Affiliated Tongji Hospital, Tongji Medical College, Huazhong Science and Technology University, Wuhan 430030, China (Tel: 86-27-83662882; Fax: 86-27-83662892; Email: xdu_f@hotmail.com) |
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Abstract BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.
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2004, Vol. 3 
