The role of proteins belonging to a plastin gene family in host defenses against bacterial infections.

属于塑性蛋白基因家族的蛋白质在宿主防御细菌感染中的作用。

• 批准号: 13670275
• 负责人: SHINOMIYA Hiroto
• 金额: $ 2.37万
• 依托单位: Ehime University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670275/
• 关键词: p65; L-plastin; phosphorylation; host defense; cytoskeleton; cell adhesion; actin; recombinant protein; immunostaining; P65

We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages. It has turned out to be a murine homologue of human L-plastin that was identified as a novel protein in human cancer cells. P65/L-plastin is characterized by a series of Ca^<2+>-, calmodulin- and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells. We have expressed a recombinant γ p65/L-plastin in E. coli that binds to b-actin and prepared high-titer antibodies using large amounts of the protein as immunogen. Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components. We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies including autoantibodies against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes.

我们以前确定了一个65 kDa的蛋白质（p65），在活化的巨噬细胞磷酸化。它已被证明是人类L-plastin的小鼠同源物，被鉴定为人类癌细胞中的新型蛋白质。P65/L-plastin具有一系列Ca^2+、钙调蛋白和肌动蛋白结合结构域，被认为在白细胞和癌细胞中起关键作用。我们在大肠杆菌中表达了重组γ p65/L-plastin。大肠杆菌中的蛋白质结合b-肌动蛋白，并使用大量的蛋白质作为免疫原制备高滴度抗体。抗rp 65/L-plastin抗体识别天然p65/L-plastin以及rp 65/L-plastin，并使我们能够检测细胞内p65/L-plastin的精细结构，并且发现其定位通过细菌组分的刺激而广泛改变。我们进一步开发了一种酶联免疫吸附测定系统和流式细胞术方法，使用这些试剂，这使得有可能测量抗体，包括对p65/L-plastin的自身抗体，并评估白细胞中蛋白质的成熟依赖性表达。

项目成果

Shinomiya H, et al.: "Detection of the activities of multiple protein kinases in lipopolysaccharide-stimulated macrophages by renaturation"J. Clin. Exp. Hematopathol. 41(2). 115-118 (2001)

Shinomiya H 等人：“通过复性检测脂多糖刺激的巨噬细胞中多种蛋白激酶的活性”J。

Hamada, K., et al.: "Molecular cloning of human squamous cell carcinoma antigen 1 gene and characterization of its promoter"Biochem. Biophys. Acta.. 1518(1-2). 124-131 (2001)

Hamada, K. 等人：“人鳞状细胞癌抗原 1 基因的分子克隆及其启动子的表征”Biochem。

Maruyama, S., et al.: "Identification of IFN regulatory facter-1 binding site in IL-12 p40 gene promoter"J. Immunoil. 170(2). 997-1001 (2003)

Maruyama, S., et al.：“IL-12 p40 基因启动子中 IFN 调节因子 1 结合位点的鉴定”J.

Shinomiya, H., et al.: "Detection of the activities of multiple protein kinases in lipopolysaccharide-stimulated macrophages by renaturation"J. Clin. Exp. Hematopathol.. 41(2). 115-118 (2001)

Shinomiya, H., et al.：“通过复性检测脂多糖刺激的巨噬细胞中多种蛋白激酶的活性”J.

四宮博人(分担): "細菌毒素ハンドブック"サイエンスフォーラム社(東京). 587 (2002)

Hiroto Shinomiya（撰稿人）：“细菌毒素手册”科学论坛出版（东京）587（2002）。