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

• 来源期刊: 中国药理学报（英文版）
• 关键词: baculoviruses prostaglandin-endoperoxide synthase non-steroidal anti-inflammatory agents,prostaglandins drug inhibition
• 年，卷（期）: 2004,（8）
• 页码范围: 1000-1006
• 页数: 7页
• 语种: 英文

摘要

AIM: 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 Spodopterafrugiperda (sf9) insect cell line by Bac-to-Bac baculovirus expression systems. Infected sr9 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. CONCLUSION: 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.