Expression and enzyme activity determination of human cyclooxygenase-1 and -2 in a baculovirus-insect cell system.
OBJECTIVE
To develop an in vitro intact cell-based assay for screening selective cyclooxygenase inhibitors.


METHODS
Human cyclooxygenase-1 (hCOX-1) and cyclooxygenase-2 (hCOX-2) genes were cloned from human monocyte cell line THP-1 cells and expressed in Spodoptera frugiperda (sf9) insect cell line by Bac-to-Bac baculovirus expression systems. Infected sf9 cells were harvested 24 h post-infection (hpi), and distributed to a 24-well plate, preincubated with various nonsteroidal anti-inflammatory drugs, and challenged with 10 mmol/L arachidonic acid; the cyclooxygenase activity was assessed indirectly by prostaglandin E2-specific radioimmunoassay.


RESULTS
Polymerase chain reaction detection demonstrated that hCOX-1 and hCOX-2 were transposed to the bacmid. Western blot analysis showed that infected sf9 cells could express hCOX-1 and hCOX-2 proteins. Radioimmunoassay demonstrated that both recombinant proteins functioned well in sf9 cells.


CONCLUSIONS
Human cyclooxygenase-1 and cyclooxygenase-2 were successfully expressed in sf9 insect cell line. It can be utilized for the identification of potent and selective inhibitors of hCOX-1 and/or hCOX-2.
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Wei-Yu
WY
Zhang
Xin-Ning
XN
Yang
Dao-Zhong
DZ
Jin
Xin-Zu
XZ
Zhu
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D016367:Baculoviridae / Q000235:genetics*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003001:Cloning, Molecular; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D007951:Leukemia, Myeloid / Q000235:genetics / Q000473:pathology; D008565:Membrane Proteins; D009578:Nitrobenzenes / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D013449:Sulfonamides / Q000494:pharmacology
D000894:Anti-Inflammatory Agents, Non-Steroidal; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D009578:Nitrobenzenes; D011994:Recombinant Proteins; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D007213:Indomethacin