Davis F M, Tsao T Y, Fowler S K, Rao P N
Proc Natl Acad Sci U S A. 1983 May;80(10):2926-30. doi: 10.1073/pnas.80.10.2926.
Certain proteins or activities are present in mitotic cells but not in interphase cells. These proteins may be synthesized or activated, or both, just prior to mitosis and are responsible for the breakdown of the nuclear envelope and the condensation of chromosomes. To learn more about the nature of these proteins, we raised monoclonal antibodies to mitotic cells. Spleen cells from mice immunized with a 0.15 M NaCl extract of synchronized mitotic HeLa cells were fused with SP2/0-Ag14 mouse myeloma cells, and hybrids were selected in medium containing hypoxanthine, methotrexate, thymidine, and glycine. Two different hybridoma clones secreting antibodies reactive with mitotic and meiotic cells from every species tested were isolated. Chromosomes as well as cytoplasm in mitotic cells reacted with the antibodies, as detected by indirect immunofluorescence. The proteins from mitotic cells were separated by electrophoresis in NaDodSO4/polyacrylamide slab gels, transferred to nitrocellulose sheets, and stained immunochemically. The two antibodies, designated MPM-1 and MPM-2, recognize a family of polypeptides with apparent molecular masses of 0.40 to greater than 200 kilodaltons (kDa). Both antibodies reacted strongly with three polypeptide bands of 182 kDa, 118 kDa, and 70 kDa. Only mitotic cells exhibited the protein bands that were recognized by the antibodies. All these bands were found to be phosphoproteins as shown by 32P labeling and autoradiography and their removal by alkaline phosphatase treatment.
某些蛋白质或活性物质存在于有丝分裂细胞中，而在间期细胞中不存在。这些蛋白质可能在有丝分裂前刚刚合成或被激活，或者两者皆有，它们负责核膜的解体和染色体的凝聚。为了更多地了解这些蛋白质的性质，我们制备了针对有丝分裂细胞的单克隆抗体。用同步化的有丝分裂期海拉细胞的0.15 M NaCl提取物免疫的小鼠脾细胞与SP2/0-Ag14小鼠骨髓瘤细胞融合，并在含有次黄嘌呤、甲氨蝶呤、胸腺嘧啶核苷和甘氨酸的培养基中筛选杂交细胞。分离出了两个不同的杂交瘤克隆，它们分泌的抗体能与所测试的每个物种的有丝分裂和减数分裂细胞发生反应。通过间接免疫荧光检测发现，有丝分裂细胞中的染色体以及细胞质都与这些抗体发生反应。有丝分裂细胞中的蛋白质在NaDodSO4/聚丙烯酰胺平板凝胶中进行电泳分离，转移到硝酸纤维素膜上，然后进行免疫化学染色。这两种抗体，分别命名为MPM-1和MPM-2，识别一组表观分子量为0.40至大于200千道尔顿（kDa）的多肽。两种抗体都与182 kDa、118 kDa和70 kDa的三条多肽带强烈反应。只有有丝分裂细胞显示出能被抗体识别的蛋白带。如32P标记和放射自显影所示，所有这些条带都是磷蛋白，并且通过碱性磷酸酶处理可以去除它们。
