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.

我们先前鉴定出一种65 kDa蛋白（p65），该蛋白在活化的巨噬细胞中被磷酸化。事实证明，它是人类L-普拉斯汀的鼠类同源物，在人类癌细胞中被鉴定为一种新型蛋白质。 p65/l-普拉斯汀的特征是一系列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着至关重要的作用。我们已经在大肠杆菌中表达了一种与B-肌动蛋白结合并使用大量蛋白作为免疫原的重组γp65/l-普拉斯汀，并制备了高滴度抗体。抗RP65/L-普拉斯汀抗体识别天然p65/l-普拉斯汀以及RP65/L-普拉斯汀，使我们能够检测细胞内p65/l-plastin的细胞结构，并且建立在细菌成分的刺激中，其本地化是广泛改变的。我们进一步开发了一种使用这些试剂的酶联免疫吸附测定系统和流式细胞术方法，这使得可以测量抗体，包括针对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。

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.

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。

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）。

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.