Gene cloning and expression of arachidonate 12-lipoxygenase

用抗花生四烯酸12-脂氧基酶的单抗，用免疫亲和层析法纯化了猪白细胞中的花生四烯酸12-脂氧基酶。纯化的酶被赖氨酰内肽酶消化，该酶特异性地裂解赖氨酸残基的羧基一侧。用气相测序仪测定了酶的多肽片段和N末端的氨基酸序列。根据多肽的部分氨基酸序列合成了两个寡核苷酸探针，并将其用作探针进行筛选。从猪白细胞CDNA文库中克隆了12-脂氧合酶CDNA。12-脂氧合酶基因编码663个氨基酸，分子量为74,911。用琼脂糖凝胶电泳法提取猪各组织总RNA，进行Northern印迹分析。到目前为止，该酶在白细胞中的表达最多，其次是垂体、肺、空肠和脾，这表明该酶在猪的各种组织中都有分布。为了在大肠杆菌中表达12-脂氧合酶基因，构建了含有tac启动子的PKK表达载体。用重组表达载体转化大肠杆菌，加入异丙硫基-β-D-半乳糖苷诱导表达12-脂氧合酶蛋白。转化大肠杆菌提取液与花生四烯酸孵育后，经反相高效液相色谱鉴定为12-羟基二十碳四烯酸。结果表明，在大肠杆菌中表达的酶具有12-脂氧合酶活性。此外，用抗12-脂氧合酶抗体进行Western印迹分析，在大肠杆菌提取物中检测到一条阳性条带，证实了该酶蛋白的表达。

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.