Jia Yanjun, Asai Azusa, Sakabe Isamu, Maruyama Mitsuo
Department of Mechanism of Aging Research Institute, National Center for Geriatrics and Gerontology, Obu-city, Aichi, Japan.
Hybridoma (Larchmt). 2010 Jun;29(3):205-9. doi: 10.1089/hyb.2009.0102.
We recently isolated and identified Zizimin2 as a functional factor that is highly expressed in murine splenic germinal center B cell after immunization with T-cell-dependent antigen. Zizimin2 was revealed that it is a new family member of Dock (dedicator of cytokinesis), Dock11, which was the guanine nucleotide exchange factor for Cdc42, the Rho family of low molecular weight GTPases. However, neither the molecular function of Zizimin2 in the acquired immunity nor the physiological role in decrease of immunological activity along with aging has been known. To facilitate the further analysis of physiological function of this molecule, we have established rat monoclonal antibodies (MAbs) against mouse Zizimin2 protein. Recombinant His-tagged partial mouse Zizimin2 protein was expressed in Escherichia coli, purified, and applied as an antigen for immunization. The desired hybridomas were selected by the recombinant Zizimin2 protein coated enzyme-linked immunosorbent assay screening, and we generated stable hybridoma cell lines that produced the antibody against murine Zizimin2 protein. We determined their isotypes and further examined capability or availability in immunoblotting, immunoprecipitation, or immunofluorescence microscopy. Here we demonstrated the several appropriate antibodies for immunoblotting and immunofluorescence microscopy. These MAbs should therefore be very useful tools for study of the characterization of the Zizimin2 protein and the understanding of the biological function of Zizimin2-mediated immunosenescence.
我们最近分离并鉴定了Zizimin2，它是一种功能因子，在用T细胞依赖性抗原免疫后在小鼠脾脏生发中心B细胞中高表达。研究发现Zizimin2是Dock（胞质分裂 dedicator）家族的新成员Dock11，Dock11是低分子量GTP酶Rho家族的Cdc42的鸟嘌呤核苷酸交换因子。然而，Zizimin2在获得性免疫中的分子功能以及随着衰老免疫活性降低过程中的生理作用尚不清楚。为了便于进一步分析该分子的生理功能，我们制备了针对小鼠Zizimin2蛋白的大鼠单克隆抗体（MAbs）。重组His标签的部分小鼠Zizimin2蛋白在大肠杆菌中表达、纯化，并用作免疫原。通过重组Zizimin2蛋白包被的酶联免疫吸附测定筛选出所需的杂交瘤，我们建立了产生抗小鼠Zizimin2蛋白抗体的稳定杂交瘤细胞系。我们确定了它们的亚型，并进一步检测了它们在免疫印迹、免疫沉淀或免疫荧光显微镜检查中的能力或可用性。在这里，我们展示了几种适用于免疫印迹和免疫荧光显微镜检查的抗体。因此，这些单克隆抗体对于研究Zizimin2蛋白的特性以及理解Zizimin2介导的免疫衰老的生物学功能应该是非常有用的工具。
