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目的 明确 5C5蛋白为分化抗原 ,检测其在人免疫细胞的表达 ,以及它的信号传导作用。方法 用 5C5蛋白免疫小鼠 ,制备抗 5C5蛋白的单抗。用反义技术制备 5C5蛋白表达抑制的 3D5细胞。用免疫荧光染色和Western印迹确定 5C5单抗的特异性。用胞膜蛋白免疫沉淀和流式细胞术确定其分化抗原的性质。用双色免疫荧光染色流式细胞术检测 5C5分化抗原在多种人免疫细胞的表达。用Fura 3 AM试剂作探针 ,用激光共聚焦显微镜观察胞内Ca2 + 浓度的变化。结果 制备了抗 5C5蛋白的特异性单抗。用 5C5单抗作探针 ,从 3D5细胞膜免疫沉淀到 1条相对分子质量 (Mr)为 19× 10 3的蛋白带 ,并在活 3D5细胞见阳性免疫荧光染色。 5C5分化抗原在人周围血CD19+ 、CD14 + 、CD4 + 、CD8+ 和CD5 6 + 细胞的表达率分别为 (33.4 7± 7.9) %、(86 .13± 6 .7) %、(15 .79± 7.1) %、(18.11±12 .77) %和 (11.76± 7.4 7) % ,在人免疫细胞株的表达率分别为 5 8.0 % (Nalm6 )、72 .7% (3D5 )、5 3.6 %(BJAB)、6 1.0 % (Daudi)、6 .9% (SKW6 )、6 .3% (Jurkat)、5 .8% (Peer)、2 1.3% (K5 6 2 )和 39.1% (U937)。3D5细胞在 5C5单抗刺激下 ,胞内Ca2 + 浓度逐渐增高 ,可达 2倍左右 ,对照小鼠Ig则无此作用。结论5C5蛋白为一个分化抗原 ,在人周?
Objective To clarify the expression of 5C5 protein in human immune cells and its signal transduction. Methods Mice were immunized with 5C5 protein to prepare monoclonal antibodies against 5C5 protein. 3D5 cells with suppressed expression of 5C5 protein were prepared using antisense technology. Specificity of the 5C5 mAb was confirmed by immunofluorescence staining and Western blotting. Cell membrane protein immunoprecipitation and flow cytometry were used to determine the nature of the differentiated antigen. Two-color immunofluorescence staining was used to detect the expression of 5C5 differentiated antigen in a variety of human immune cells. The Fura 3 AM reagent was used as a probe, and the change of intracellular Ca2 + concentration was observed by laser confocal microscopy. Results Specific monoclonal antibodies against 5C5 protein were prepared. A 5C5 monoclonal antibody was used as a probe to immunoprecipitate a protein band with a molecular mass of 19 × 10 3 from 3D5 cell membrane and positive immunofluorescence staining in viable 3D5 cells. The expression rates of 5C5 differentiated antigen in peripheral blood were (33.4 7 ± 7.9)%, (86.13 ± 6.7)%, (15.79 ± 7.1%), (18.11 ± 12.77)%, and (11.76 ± 7.47)%, respectively. The expression rates in human immune cell lines were 58.0% (Nalm6), 72.7% (3D5) and 53.6% BJAB, 6 1.0% (Daudi), 6.9% (SKW6), 6.3% (Jurkat), 5.8% (Peer), 2.1.3% (K5 6 2) and 39.1% (U937). 3D5 cells stimulated by 5C5 mAb, intracellular Ca2 + concentration gradually increased up to 2 times the control mouse Ig is no such effect. Conclusion 5C5 protein is a differentiated antigen in human weeks?