Expression and enzyme activity determination of human cyclooxygenase-1 and -2 in a baculovirus-insect cell system

Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Acta Pharmacologica Sinica (Impact Factor: 2.91). 09/2004; 25(8):1000-6.
Source: PubMed


To develop an in vitro intact cell-based assay for screening selective cyclooxygenase inhibitors.
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.
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.
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.

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