Abstract:

Objective To construct the eukaryotic expression vector of heme oxygenase-1 (HO-l) gene, and transfect the recombinant expression vector to rat peritoneal mesothelial cells (RPMCs) in vitro. Methods Total RNA was extracted from SD rat spleen, and the full length fragment of HO-1 CDS part was amplified by RT-PCR. The product of PCR was cloned to pMD-18T vector. The pMD-18T-HO-1 and pcDNA3-1 (－) vector were digested with restriction endonuclease BamHⅠ and KpnⅠ, then T4 DNA ligase was used to ligate HO-1 gene fragment and digested pcDNA3-1 (－) vector to construct the eukaryotic expression vector pcDNA3-1 (－)-HO-1. The recombinant plasmid pcDNA3-1 (－)-HO-1 was transfected into RPMCs. The expression of HO-1 in RPMCs was detected by RT-PCR and Western blotting respectively. Results The full length of rat HO-1 gene was cloned, and the eukaryotic expression vector pcDNA3-1 (－)-HO-1 was successfully constructed. Restriction endonucleases digestion confirmed the target DNA fragment was inserted into the expression vector, and was verified by DNA sequencing. RT-PCR and Western blotting revealed that HO-1 was highly expressed in RPMCs after transfection with pcDNA3-HO-1. Conclusion HO-1 eukaryotic expression vector has been successfully constructed, and HO-1 expresses effectively in RPMCs, which lays a foundation for the further study of the effect of HO-1 in RPMCs injury during peritoneal dialysis.

Key words: heme oxygenase-1, expression vector, peritoneal mesothelial cell, rat