Yu Min, Wang Zehua, Gong Junli, Ma Mingxing, Jiao Yang, Huang Weiwei, Lü Qi, Li Lin, Yang Hui, Tan Deyong
Laboratory of Biochemistry and Molecular Biology, School ofLife Science, Yunnan University, Kunming 650091, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Jan;26(1):57-62.
Human arrest defective 1(hARD1) is an acetyltransferase catalyzing the N-terminal acetylation of proteins after translation. The high expression of hARD1 could be an indicator of the breast cancer. In current study, we produced an anti-hARD lp monoclonal antibody that could specifically recognize ARD1 in breast cancer tissues by using the immunohistochemical assay. The full-length His-tag hARD1 protein (1-235 aa) was over-expressed in Escherichia coli, and purified recombinant protein was injected into Balb/c mice to perform immunization procedure. Eight stable positive monoclonal cell lines were isolated. ELISA results demonstrated that all light chains of antibodies were kappa, and the heavy chains displayed three subtypes IgG1, IgG2a and IgG2b, respectively. A monoclonal antibody, which could specifically recognize hARD1 protein in breast cancer tissues, was identified by screening different cancer tissues using antibody-specificity method. Further, the specificity of the antibody was confirmed by Western blotting analysis. Our study would facilitate breast cancer diagnosis by using this ARD1 monoclonal antibody in clinic. Also, this antibody could be used as an important tool for further investigating the role of ARD1 in tumorigenesis.
人类 Arrest 缺陷 1（hARD1）是一种乙酰转移酶，可在翻译后催化蛋白质的 N 端乙酰化。hARD1 的高表达可能是乳腺癌的一个指标。在当前研究中，我们制备了一种抗 hARD1 单克隆抗体，通过免疫组织化学分析，该抗体能够特异性识别乳腺癌组织中的 ARD1。全长 His 标签的 hARD1 蛋白（1 - 235 个氨基酸）在大肠杆菌中过表达，纯化后的重组蛋白注射到 Balb/c 小鼠体内进行免疫程序。分离出了八个稳定的阳性单克隆细胞系。ELISA 结果表明，所有抗体的轻链均为 κ 链，重链分别呈现三种亚型 IgG1、IgG2a 和 IgG2b。通过抗体特异性方法筛选不同的癌组织，鉴定出一种能够特异性识别乳腺癌组织中 hARD1 蛋白的单克隆抗体。此外，通过 Western 印迹分析证实了该抗体的特异性。我们的研究将有助于在临床中使用这种 ARD1 单克隆抗体进行乳腺癌诊断。而且，该抗体可作为进一步研究 ARD1 在肿瘤发生中作用的重要工具。
