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


CONCLUSIONS
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.
Institute of Organ Transplantation, Affiliated Tongji Hospital, Tongji Medical College, Huazhong Science and Technology University, Wuhan 430030, China. xdu_f@hotmail.com
Dun-Feng
DF
Du
Sheng
S
Chang
Bi-Cheng
BC
Chen
Zhong-Hua
ZH
Chen
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics*; D051547:Heme Oxygenase-1; D010957:Plasmids / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000235:genetics; D013154:Spleen / Q000502:physiology; D014162:Transfection
D018076:DNA, Complementary; D011994:Recombinant Proteins; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1