蛋白酶活化受体-1单克隆抗体的制备与鉴定

发布时间：2018-12-15 18:53

【摘要】： 背景与目的：蛋白酶活化受体-1(PAR-1)是一种的蛋白质，由425个氨基酸组成，含7个疏水跨膜域，为典型的G蛋白偶联受体。PAR-1广泛分布在各种组织细胞上，包括上皮细胞，血小板，内皮细胞，成纤维细胞，单核细胞，T淋巴细胞，成骨细胞，平滑肌细胞，神经细胞，神经胶质细胞及某些癌细胞系等。PAR-1具有广泛的生物学效应，参与诸多疾病的发生发展，因此，，我们制备廉价、有效的抗PAR-1mAb具有重要意义。 方法：以PAR-1特异性片段为免疫原(13肽)免疫BALB／c小鼠，取免疫小鼠脾细胞和小鼠骨髓瘤NS-1细胞融合。通过间接ELISA法筛选可特异性分泌抗PAR-1 mAb的杂交瘤细胞。采用capture ELISA法鉴定mAb的Ig亚类；通过ELISA、Dot blot、免疫组织化学染色法、流式细胞分析、激光共聚焦扫描显微镜技术鉴定mAb的特异性。 结果：获得2株可稳定分泌抗PAR-1 mAb的杂交瘤细胞，其亚型分别为IgM，IgG2b。Dot blot检验表明，mAbs可与膜上的抗原结合。免疫组织化学染色表明，mAb与人扁桃体、结肠组织中的淋巴细胞、包皮组织中的成纤维细胞、肺组织中的巨噬细胞反应呈阳性。流式细胞仪分析显示，2株mAb与人肺腺癌细胞系A549细胞膜上及细胞胞浆内的受体均呈阳性反应。激光共聚焦扫描显微镜观察，A549细胞的胞膜及胞浆内均有荧光标记的阳性反应物。 结论：成功地制备抗PAR-1 mAb，为变态反应性疾病，炎症性疾病及其他疾病的研究工作提供了有用的试剂。
[Abstract]:Background & objective: protease activated receptor-1 (PAR-1) is a kind of protein consisting of 425 amino acids and contains 7 hydrophobic transmembrane domains. PAR-1 is a typical G-protein-coupled receptor. PAR-1 is widely distributed in various tissues and cells. Including epithelial cells, platelets, endothelial cells, fibroblasts, monocytes, T lymphocytes, osteoblasts, smooth muscle cells, nerve cells, PAR-1 has a wide range of biological effects and participates in the occurrence and development of many diseases. Therefore, it is of great significance to prepare cheap and effective anti-PAR-1mAb. Methods: BALB/c mice were immunized with PAR-1 specific fragment (13 peptide). Spleen cells of mice were fused with NS-1 cells of myeloma mice. Hybridoma cells secreting anti-PAR-1 mAb were screened by indirect ELISA method. The Ig subclasses of mAb were identified by capture ELISA method, and the specificity of mAb was identified by ELISA,Dot blot, immunohistochemical staining, flow cytometry and laser confocal scanning microscopy. Results: two hybridoma cells stably secreting anti PAR-1 mAb were obtained. The subtypes of these hybridomas were IgM,IgG2b.Dot blot test. The results showed that mAbs could bind to the antigens on the membrane. Immunohistochemical staining showed that mAb reacted positively with lymphocytes in human tonsil, colon, fibroblasts in prepuce and macrophages in lung tissue. The results of flow cytometry showed that both mAb and A549 cells showed positive reaction to the receptors on the cell membrane and in the cytoplasm of A549 cell line. Fluorescence labeled positive reactants were observed in the membrane and cytoplasm of A549 cells by confocal laser scanning microscopy. Conclusion: the successful preparation of anti-PAR-1 mAb, may provide useful reagents for the research of allergic diseases, inflammatory diseases and other diseases.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【参考文献】

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1 王海燕,何韶衡,郑燕珊;蛋白酶激活受体1介导人肺上皮细胞分泌MCP-1[J];第三军医大学学报;2005年17期

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