抗人分泌性白细胞蛋白酶抑制剂和白介素-28A单克隆抗体的制备与鉴定

发布时间：2018-09-01 18:36

【摘要】：背景与目的：人分泌性白细胞蛋白酶抑制剂(human secretory leukocyte protease inhibitor，hSLPI)是一种低分子量的嗜弹性蛋白酶抑制剂，为粘膜分泌的非糖基化蛋白。SLPI来源于上皮细胞、肥大细胞、中性粒细胞、单核细胞和巨噬细胞，广泛分布在皮肤、呼吸道、胃肠道、唾液腺和生殖腺。SLPI能抑制中性粒细胞的弹性蛋白酶、组织蛋白酶G、肥大细胞的类糜蛋白酶、胰蛋白酶和糜蛋白酶，在变态反应性疾病和炎症中起重要作用。检测SLPI的水平有利于深入了解在变态反应性疾病和粘膜炎症发病机制中，蛋白酶和蛋白酶抑制剂平衡的问题。因此，制备廉价、有效的抗hSLPI单克隆抗体(mAb)具有重要意义。方法：以hSLPI为免疫原免疫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hSLPI mAb的杂交瘤细胞。采用Ig类与亚类鉴定试剂盒鉴定mAb的Ig亚类；通过ELISA、Western 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10株可稳定分泌抗hSLPI mAb的杂交瘤细胞，其分泌的mAb均为IgM。Western blot分析表明，此mAb所识别的靶分子的相对分子质量(Mr)约为12000。免疫组织化学染色表明，mAb与肺、结肠及包皮组织中的上皮细胞、疑似肥大细胞，扁桃体组织中的疑似肥大细胞的反应性呈阳性。流式细胞仪分析显示，10株mAb与人肺腺癌细胞系A549细胞中的SLPI均呈阳性反应。激光共聚焦扫描显微镜观察，荧光标记的阳性反应物主要位于A549细胞的胞浆内。结论：成功地制备抗hSLPI mAb，为变态反应性疾病和炎症性疾病的研究工作提供了有用的试剂。
[Abstract]:Background & AIM: human secretory leukocyte protease inhibitor (human secretory leukocyte protease inhibitor,hSLPI) is a low molecular weight elastase inhibitor. Monocytes and macrophages, widely distributed in the skin, respiratory tract, gastrointestinal tract, salivary gland and gonad. SLPI inhibits neutrophil elastase, cathepsin G, mast cell chymoid, trypsin and chymotrypsin. Play an important role in allergic diseases and inflammation. The level of SLPI is helpful to understand the balance of protease and protease inhibitor in the pathogenesis of allergic diseases and mucosal inflammation. Therefore, the preparation of cheap and effective anti-hSLPI monoclonal antibody (mAb) is of great significance. Methods: BALB/c mice were immunized with hSLPI. Spleen cells of mice were fused with NS-1 cells of myeloma mice. Hybridoma cells secreting anti-hSLPI mAb were screened by indirect ELISA method. The Ig subclasses of mAb were identified by Ig and subclass identification kit, and the specificity of mAb was identified by ELISA,Western blot, immunohistochemical staining, flow cytometry and confocal laser scanning microscopy. Results: ten hybridoma cells stably secreting anti hSLPI mAb were obtained. The mAb secreted by the hybridoma cells were all analyzed by IgM.Western blot. The relative molecular weight (Mr) of the target molecule recognized by the mAb was about 12 000. Immunohistochemical staining showed that mAb was positive for the reactivity of epithelial cells in lung, colon and prepuce tissues, suspected mast cells, and suspected mast cells in tonsils. Flow cytometry analysis showed that 10 mAb strains were positive for SLPI in human lung adenocarcinoma cell line A549. The fluorescence labeled positive reactants were mainly located in the cytoplasm of A549 cells under confocal laser scanning microscope. Conclusion: the successful preparation of anti-hSLPI mAb, provides a useful reagent for the study of allergic and inflammatory diseases.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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