依赖于环腺苷酸蛋白激酶的单克隆抗体制备及其在正常和恶性细胞内的免疫细胞化学定位观察

用依赖于环腺苷酸(cAMP)的蛋白激酶(蛋白激酶A,PKA)免疫LOU/C大鼠,分离出脾细胞与大鼠骨髓瘤细胞系IR 983 F融合,经HAT培养液筛选和ELISA检测,获得1株分泌抗PKA单克隆抗体的大鼠杂交瘤细胞系B5 A8。免疫扩散实验表明,该单克隆抗体亚类为IgG_1。免疫印迹实验显示,此单克隆抗体可识别蛋白激酶A的52～56 kd多肽。利用经亲和层析法纯化的单克隆抗体,采用免疫荧光细胞化学方法检测人正常成纤维细胞、人胃癌细胞系和小鼠艾氏腹水癌细胞内的蛋白激酶A定位,结果显示蛋白激酶A主要定位于细胞质,与细胞骨架的分布一致。同步化的胃癌细胞系蟹白激酶A在G_2期进入胞核,经外源性cAMP处理后的胃癌细胞蛋白激酶A向核周围积聚。本文对蛋白激酶A在细胞内分布的变化进行了讨论。

PREPARATION OF MONOCLONAL ANTIBODY AGAINST PROTEIN KINASE A AND ITS IMMUNOCYTOCHEMICAL LOCALIZATION IN NORMAL AND MALIGNANT CELLS

A rat monoclonal antibody of the IgG1 class, McAb B5A8, specific for the cAMP-dependent protein kinase(PKA) was produced. Western blot analysis revealed specific binding of the antibody to protein of 52-56 kd.The affinity-purified McAb BSA8 was labeled with FITC. Immunofluorescent localization of PKA was examined in human fibroblasts, gastric cancer cell line (MGc 80-3)and EAC cells. The distribution of PKA on the cytoplasmic microtubule network, Golgi region and nucleoli were observed. PKA was localized in the nuclear region in G2 phase of synchronized MGc 80-3 cells, and it was only found around the nuclei of MGc 80-3 cells which were incubated with DBcAMP. The changes in the distribution of PKA in cells are discussed.