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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。
LOU/C rats were immunized with a protein kinase (PKA) dependent on cyclic adenosine monophosphate (cAMP) and spleen cells were isolated and fused with rat myeloma cell line IR 983 F and screened by HAT medium and ELISA. A rat hybridoma cell line B5 A8 secreting an anti-PKA monoclonal antibody was obtained. The immunodiffusion experiment showed that the monoclonal antibody subclass is IgG_1. Western blotting experiments showed that this monoclonal antibody can recognize the 52-56 kd polypeptide of protein kinase A. Using monoclonal antibodies purified by affinity chromatography, immunofluorescence cytochemistry was used to detect protein kinase A localization in human normal fibroblasts, human gastric cancer cell lines and Ehrlich ascites carcinoma cells. Mainly located in the cytoplasm, consistent with the distribution of the cytoskeleton. The synchronized gastric cancer cell line Crabskin kinase A entered the nucleus in the G2 phase, and the exogenous cAMP-treated gastric cancer cell protein kinase A accumulated around the nucleus. This article discusses the changes in the distribution of protein kinase A in cells.