中文摘要：

目的人单酰基甘油脂肪酶（humanmonoacylglycerol lipase，MAGL）是一类促进脂肪分解成甘油和脂肪酸的丝氨酸水解酶，是抗肿瘤药物研发的新靶点。为了建立高通量的MAGL抑制剂筛选模型，本研究对人MAGL进行了基因克隆、体外重组表达、纯化以及活性研究。方法通过RT-PCR方法从人脑总RNA中扩增人MAOL基因，并将该基因克隆．NpET28a（＋）表达载体。将重组蛋白在E-coliBL21（DE3）宿主菌中通过1mmol／LIPTG诱导表达。表达产物用Ni．NTA琼脂糖亲和层析柱进行分离纯化，通过SDS—PAGE进行分析。以7-hydroxycoumarinylarachidonate（7-HAC）为荧光底物进行酶的活性测定。结果成功扩增出人MAGL基因，并构建TpET28a．MAGL表达载体。SDS—PAGE检测结果表明重组人MAGL蛋白在宿主菌中的表达量可达菌体总蛋白量的25％，经Ni-NTA琼脂糖亲和层析柱分离纯化后的重组蛋白纯度大于90％，其比活力达到7900IU／mg，较纯化前提高了23倍。酶活性分析结果证明重组表达并纯化的蛋白具有单酰基甘油脂肪酶活性，另外，利用己知的MAGL抑制剂N．arachidonylmaleimide（NAM）对该酶表现出强的抑制活性，IC50值为0．53μmol／L。结论本研究成功利用大肠埃希菌原核表达体系重组表达并纯化了人单酰基甘油脂肪酶，为建立以MAGL为靶点的抗肿瘤药物筛选模型奠定了基础。

英文摘要：

Objective Human monoacylglycerol lipase （MAGL）, a serine hydrolase that converts monoglycerides to fatty acid and glycerol, plays crucial roles in cancer pathogenesis, and has become a novel drug target for antitumor drug development. In present study, to develop a high throughput screening asaay for MAGL inhibitor, the human MAGL was recombinantly expressed in E. coli. Methods The human MAGL gene was cloned by reverse transcription-PCR（RT-PCR） from human brain total RNA, and was inserted into expression vector pET28a（＋）. The recombinant MAGL protein was expressed in E. coli host strain BL21（DE3） with lmM IPTG induction, and purified by Ni-NTA affinity chromotagraphy. Monoacylglycerol lipase activity was determined by a fluorescence-based assay using 7-hydroxycoumarinyl arachidonate as substrate. Results The human MAGL gene was successfully amplified from human brain total RNA and expressed in E. coli BL21 （DE3）. The recombinant human MAGL （rhMAGL） could account for approximately 25% of total proteins by SDS-PAGE analysis. After purified by Ni-NTA affinity chromotagraphy, the purity of the rhMAGL was over 90%. The specific activity of the purified rhMAGL is 7900 units/mg protein with 23 fold increase over the crude extract.The rhMAGL was proved to prossess monoacylglycerol hydrolase activity. In addition, N-Arachidonyl Maleimide, a known MAGL inhibitor, could significantly inhibit the rhMAGL activity with an IC5o value of 0.53gmol/L. Conclusion Human MAGL was successfully recombinantly expressed and purified, which laid the basis for screening human MAGL inhibitors in new antitumor drug development.