Gene cloning and expression of arachidonate 12-lipoxygenase

花生四烯酸12-脂氧合酶基因克隆及表达

基本信息

  • 批准号:
    02670110
  • 负责人:
  • 金额:
    $ 1.41万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)
  • 财政年份:
    1990
  • 资助国家:
    日本
  • 起止时间:
    1990 至 1991
  • 项目状态:
    已结题

项目摘要

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.
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。

项目成果

期刊论文数量(29)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Yodhimoto,T.: "Molecular cloning and expression of human arachidonate 12ーlipoxygenase" Biochem.Biophys.Res.Commun.172. 1230-1235 (1990)
Yodhimoto, T.:“人花生四烯酸 12-脂氧合酶的分子克隆和表达”Biochem.Biophys.Res.Commun.172(1990)。
  • DOI:
  • 发表时间:
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    0
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  • 通讯作者:
Ueda,N.: "Localization of arachidonate 12ーlipoxygease in parenchymal cells of porcine pituitary" J.Biol.Chem.265. 2311-2316 (1990)
Ueda, N.:“花生四烯酸 12-脂氧酶在猪垂体实质细胞中的定位”J.Biol.Chem.265 (1990)。
  • DOI:
  • 发表时间:
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    0
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  • 通讯作者:
Ueda,N.: "Localization of arachidonate 12ーlipoxygenase in parenchymal cells of porcine pituitary" J.Biol.Chem.265. 2311-2316 (1990)
Ueda, N.:“花生四烯酸 12-脂氧合酶在猪垂体实质细胞中的定位”J.Biol.Chem.265 (1990)。
  • DOI:
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  • 影响因子:
    0
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  • 通讯作者:
Matsuda, S.: "Analysis of non-heme iron in arachidonate 12-lipoxygenase of porcine leukocytes." Biochem. Biophys. Acta. 1084. 202-204 (1991)
Matsuda, S.:“猪白细胞花生四烯酸 12-脂氧合酶中非血红素铁的分析。”
  • DOI:
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  • 影响因子:
    0
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  • 通讯作者:
Yoshimoto,T.: "Prostaglandins and Related Compounds,Vol 21,pp.29ー32" Raven Press, 4 (1990)
Yoshimoto, T.:“前列腺素和相关化合物,第 21 卷,第 29-32 页”Raven Press,4 (1990)
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YOSHIMOTO Tanihiro其他文献

YOSHIMOTO Tanihiro的其他文献

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{{ truncateString('YOSHIMOTO Tanihiro', 18)}}的其他基金

Pathophysiological role of lipid molecules produced by 12/l5-lipoxygenase
12/l5-脂氧合酶产生的脂质分子的病理生理作用
  • 批准号:
    12470024
  • 财政年份:
    2000
  • 资助金额:
    $ 1.41万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Mechanisms for tissue specific expression and cell growth of 12/15-lipoxygenase
12/15-脂氧合酶的组织特异性表达和细胞生长机制
  • 批准号:
    10670134
  • 财政年份:
    1998
  • 资助金额:
    $ 1.41万
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
Studies on Arachidonate Oxygenases of Human Platelets
人血小板花生四烯酸加氧酶的研究
  • 批准号:
    63570115
  • 财政年份:
    1988
  • 资助金额:
    $ 1.41万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (C)

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    22K20838
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食品成分变化对花生四烯酸代谢酶催化活性影响的研究
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