关键词: 前列腺素E1, 环氧合酶1, 真核表达, 重组载体, 催化活性

Abstract: Objective: To construct the eukaryotic expression vector of cycxygenase 1 (COX1) and to verify that COX1 protein can catalyze the synthesis of prostaglandin E1 (PGE1) in vitro, and to find a method to efficiently obtain PGE1. Methods: The total RNA of human microvascular endothelial cells (HMECs) was extracted and reversely transcribed into cDNA, which was served as the template to amplify the human COX1 gene by PCR. After double enzyme digestion, COX1 gene was connected with the eukaryotic expression vector pCMV6 to construct the recombinant plasmid pCMV6-COX1. Bioinformatic online softwares were used to analyze the hydrophobicity, transmembrane region, signal peptide, secondary structure and tertiary structure of COX1 protein. Afterwards, the human COX1 eukaryotic expression plasmid was expressed in the HEK-293F cells by liposomes. The cells were divided into control group (the HEK-293F cells without recombinant plasmid transfection), transfection 2 d group and transfection 5 d group. Then the cells and the cell culture supernatants were collected, respectively; the expression levels of COX1 protein were detected by Western blotting method. The activities of PGE1 synthesized by dohomo-γ-linolenic acid catalyzed by the eukaryotic expression COX1 protein and crude enzyme extract from sheep seminal vesicles were compared. Results: The recombinant plasmid pCMV6-COX1 was successfully constructed and identified by PCR, double digestion and sequencing. The length of target gene was about 1 800 bp. The bioinformatics analysis results showed that COX1 protein was a water-soluble protein. The amino acids at position 1-53 were signal peptides, the amino acids at position 34-53 were transmembrane regions; there were 36 serine phosphorylation sites, 14 threonine phosphorylation sites and 9 tyrosine phosphorylation sites. The prediction of secondary structure showed that the ratio of irregular crimp, alpha helix, extension chain and β angle were 46.20%, 41.03%, 10.18% and 2.58%, respectively.The Western blotting results showed that the recombinant plasmid was successfully transfected into the HEK-293F cells; compared with transfection 2 d group,the expression level of COX1 protein in the supernatants of the cells in transfetion 5 d group was significantly increased(P<0.05) and the relative molecular weight of protein was 70 000. When the substrate concentration was 1%, the yield of PGE1 synthesized by COX1 protein was (50.01±1.37) ng·L^-1, which was equivalent to (90.3±0.06)% of the activity of crude enzyme extracted from 5 sheep seminal vesicles. Conclusion: The COX1 protein constructed and expressed by genetic engineering technology can catalyze the synthesis of PGE1 and meet the clinical requirements.

Key words: prostaglandin E1, cycxygenase 1, eukaryotic expression, recombinant vector, catalytic activity