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

我们以前发现了一个65 kDa的蛋白(P65)，它在激活的巨噬细胞中被磷酸化。事实证明，它是人类L的小鼠同源物-纤溶酶原蛋白被确定为人类癌细胞中的一种新蛋白质。P65/L-纤溶酶原蛋白由一系列钙调素、钙调蛋白和肌动蛋白结合区组成，被认为在白细胞和癌细胞中发挥重要作用。我们在大肠杆菌中表达了与b-肌动蛋白结合的重组γp65/L-纤溶酶原蛋白，并以其为免疫原制备了高效价抗体。抗rp65/L-纤溶酶原蛋白抗体识别天然p65/L-纤溶酶和rp65/L-纤溶酶，使我们能够检测到细胞内p65/L-纤溶酶的精细结构，并发现其定位在细菌成分的刺激下发生了很大的变化。我们进一步发展了一种酶联免疫吸附分析系统和一种使用这些试剂的流式细胞术方法，使我们能够检测包括抗p65/L-纤溶酶原蛋白的自身抗体在内的抗体，并评估该蛋白在白细胞中的成熟依赖性表达。

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.