Gene cloning and expression of arachidonate 12-lipoxygenase

用抗花生四烯酸12-脂氧化酶单克隆抗体免疫亲和层析纯化猪白细胞花生四烯酸12-脂氧化酶。用赖氨酰内肽酶消化纯化的酶，所述赖氨酰内肽酶特异性地切割赖氨酸残基的羧基侧。使用气相测序仪测定肽片段和酶的N-末端的氨基酸序列。基于肽的部分氨基酸序列合成两种寡核苷酸探针，并用作筛选探针。12-从猪白细胞cDNA文库中克隆了脂氧合酶cDNA。该基因编码663个氨基酸，分子量为74，911。用琼脂糖凝胶电泳分离猪各组织的总RNA，并进行北方印迹分析。该酶在白细胞中的表达量最大，其次是垂体、肺、空肠和脾，表明该酶在猪的多种组织中均有分布。为了在E.在pKK质粒中构建了tac启动子的表达载体。E.将重组质粒转化大肠杆菌，用异丙基硫代β-D-半乳糖苷诱导表达12-脂氧合酶蛋白。转化E.大肠杆菌与花生四烯酸一起孵育，通过反相HPLC鉴定反应产物为12-羟基-二十碳四烯酸。结果表明，在E.大肠杆菌具有12-脂氧合酶活性。用抗12-脂氧合酶抗体进行Western blot分析，在大肠杆菌中检测到一条阳性条带。大肠杆菌提取物，证实了酶蛋白的表达。

Arachidonate 12-lipoxy, enase of porcine leukocytes was purified by immunoaffinity chromato, raphy using a monoclonal antibody against the enzyme. The purified enzyme was digested by lysyl endopeptidase which specifically cleaved the carboxyl side of a lysine residue. Amino acid sequences of the peptide fragments and of N-terminus of the enzyme were determined using a gas-phase sequencer. Two oligonucleotide probes were synthesized based upon the partial amino acid sequence of the peptides, and utilized as probes for screening. 12-Lipoxygenase CDNA was cloned from a CDNA library of porcine leukocytes. The cDNA of 12-lipoxygenase encoded 663 amino acids with a calculated molecular weight of 74, 911. Total RNA of various porcine tissues was separated by agarose gel electrophoresis, and subjected to Northern blot analysis. By far the largest amount of mRNA of the enzyme was detected in leukocytes, followed by pituitary, lung, jejunum and spleen, indicating that the enzyme was distributed in a variety of porcine tissues. In order to express the cDNA of 12-lipoxygenase in E. coli, an expression vector was constructed in a pKK plasmid with tac promoter. E. coli was transformed with the recombinant plasmid, and the 12-lipoxygenase protein was induced by the addition of isopropylthio-beta-D-galactoside. The extract of the transformed E. coli was incubated with arachidonic acid, and the reaction product was identified to be 12-hydroxy-eicosatetracnoic acid as assessed by reverse-phase HPLC. The result indicated that the expressed enzyme in E. coli had 12-lipoxygenase activity. Furthermore, Western blot analysis using anti-12-lipoxygenase antibody detected a positive band in the E. coli extract, confirming the expression of the enzyme protein.