Development by Gene Engineering of Kits for Screening the Enzymes of the Arachidonate Cascade

多种生物活性化合物在称为花生四烯酸级联的代谢途径中产生。为了阐明这些化合物的生理和病理作用，必须对级联反应中的几个关键酶进行研究。这些酶也是各种药物开发的目标。本研究的目的是通过基因工程技术开发花生四烯酸级联酶的检测试剂盒。1）花生四烯酸级联酶cDNA的克隆：除了先前的5-和12-脂氧合酶、环氧合酶和白三烯A水解酶的cDNA克隆外，我们克隆了人血小板和血管内皮环加氧酶以及人血小板血栓烷A氧化酶的cDNA。2）酶cDNA的表达：5-脂氧合酶cDNA在E.大肠杆菌和酵母中的12-脂氧合酶;大肠杆菌中的血栓素A合成酶和大肠杆菌中的白三烯A水解酶。3）酶mRNA的定量测定：通过使用12-脂氧合酶cDNA作为探针，PCR扩增人血小板中的12-脂氧合酶mRNA，并以cRNA作为内标进行定量。4）花生四烯酸级联酶的酶免疫分析：建立了人血小板12-脂氧合酶的过氧化物酶联免疫分析方法，并应用于12-脂氧合酶缺乏的白血病患者。还建立了白三烯A水解酶的酶免疫分析方法。

A variety of bioactive compounds are produced in a metabolic pathway referred to as the arachidonate cascade. Several key enzymes of the cascade must be studied to elucidate the physiological and pathological roles of these compounds. These enzymes are also the targets in the development of various drugs. The purpose of this research project is the development of the assay kits for the arachidonate cascade enzymes by gene engineering techniques.1) Cloning of cDNAs for the arachidonate cascade enzymes: In addition to previous cDNA clonings for 5- and 12-lipoxygenases, cyclooxygenase and leukotriene A hydrolase, we cloned cDNAs for human platelet and vascular endothelial cyclooxygenases and for human platelet thromboxane A synthase.2) Expression of the enzyme cDNAs: 5-Lipoxygenase cDNA was expressed in E. coli and yeast, 12-lipoxygenase in E. coli, thromboxane A synthase in insect cells and leukotriene A hydrolase in E. coli.3) Quantitative assay of the enzyme mRNA: By the use of 12-lipoxygenase cDNA as a probe, the 12-lipoxygenase mRNA in human platelets was PCR-amplified and quantitated with a cRNA as an internal standard. The assay was applied to leukemia patients with reduced 12-lipoxygenase activity.4) Enzyme immunoassays for the arachidonate cascade enzymes: A peroxidase-linked immunoassay was developed for human platelet 12-lipoxygenase and applied to the leukemia patients with 12-lipoxygenase deficiency. An enzyme immunoassay was also set up for leukotriene A hydrolase.