Xiong Wei, Huang Weiwei, Jiao Yang, Ma Jie, Yu Min, Ma Mingxing, Wu Hong, Tan Deyong
Laboratory of Biochemistry and Molecular Biology, School of Life Sciences, Yunnan University, 002 Cuihu Road, Kunming 650091, PR China.
Protein Expr Purif. 2012 Mar;82(1):11-9. doi: 10.1016/j.pep.2011.10.012. Epub 2011 Nov 10.
Human mitochondrial transcription termination factor 2 (MTERF2) is a member of the mitochondrial transcription termination factors (MTERFs) family and a cell growth inhibitor. To create a specific mouse monoclonal antibody against human MTERF2, the full-length His-tag MTERF2 protein (1-385 aa) was expressed in Escherichia coli, and purified recombinant protein was injected into three BALB/c mice to perform an immunization procedure. Eight stable positive monoclonal cell lines were screened and established. ELISA results demonstrated that all antibody light chains were kappa, while the heavy chains displayed three subtypes IgG1, IgG2a, and IgG2b respectively. The sensitivity and specificity of the monoclonal antibodies against human MTERF2 were determined using immunoblotting, immunoprecipitation and immunofluorescence analyses. Furthermore, serum regulation of human MTERF2 protein expression levels in human glioma U251 cells was examined with these monoclonal antibodies and the results demonstrated that the expression level of MTERF2 protein was dramatically inhibited by the addition of serum to serum-starved cells. Taken together, our results demonstrate the functionality of these mouse anti-human MTERF2 monoclonal antibodies, which may provide a useful tool to elucidate the role of MTERF2 in human mitochondrial transcription as well as other potential activities. To our knowledge, this is the first report on the preparation and characterization of mouse monoclonal antibodies against human MTERF2.
人线粒体转录终止因子2（MTERF2）是线粒体转录终止因子（MTERFs）家族的成员之一，也是一种细胞生长抑制剂。为了制备一种针对人MTERF2的特异性小鼠单克隆抗体，将全长His标签的MTERF2蛋白（1-385个氨基酸）在大肠杆菌中表达，然后将纯化的重组蛋白注射到三只BALB/c小鼠体内进行免疫程序。筛选并建立了8个稳定的阳性单克隆细胞系。酶联免疫吸附测定（ELISA）结果表明，所有抗体的轻链均为κ链，而重链分别呈现IgG1、IgG2a和IgG2b三种亚型。使用免疫印迹、免疫沉淀和免疫荧光分析确定了针对人MTERF2的单克隆抗体的敏感性和特异性。此外，用这些单克隆抗体检测了血清对人胶质瘤U251细胞中人MTERF2蛋白表达水平的调节作用，结果表明，向血清饥饿的细胞中添加血清可显著抑制MTERF2蛋白的表达水平。综上所述，我们的结果证明了这些小鼠抗人MTERF2单克隆抗体的功能，它们可能为阐明MTERF2在人线粒体转录以及其他潜在活动中的作用提供有用的工具。据我们所知，这是关于制备和鉴定抗人MTERF2小鼠单克隆抗体的首次报道。
