抗人分泌性白细胞蛋白酶抑制剂单克隆抗体的制备和鉴定.pdf

汕头大学医学院硕士学位论文 1 抗人分泌性白细胞蛋白酶抑制剂单克隆抗体的制备与鉴定研究生: 陈玉珍导师:何韶衡摘要背景与目的: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hSLPImAb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hSLPImAb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hSLPImAb,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。关键词:人分泌性白细胞蛋白酶抑制剂;单克隆抗体;特性鉴定汕头大学医学院硕士学位论文 2 Preparationand Characterization of Monoclonal Antibodies againstHuman Secretory Leukocyte Protease Inhibitor Postgraduate: CHEN Yu-zhen Supervisor: HEShao-heng Abstract Background and objective:Human secretory leukocyte protease inhibitor(hSLPI), is a 12 kD nonglycosylatedproteinase inhibitorwith a high capacity forinhibiting neutrophil elastase (NE) present in mucous secretions. SLPI is locally produced by epithelial cells, mast cells, neutrophils, monocytes,macrophages and is widely distributed at skin, respiratory tract, gastrointestinal tract, salivary gland and genital possesses potent inhibitory activity against neutrophil elastase,cathepsin G, chymase,trypsin and chymotrypsinand play an important role in allergic and inflammatory diseases. Measuringthe levels of SLPI help us understand the protease-antiprotease balancein allergic and inflammatory diseases well. Therefore, it is meaningful to prepare and identifymonoclonal antibodies (mAbs) against human secretory leukocyte protease inhibitor. Methods:BALB/cmice were immunized with hSLPI, and hybridoma cell lines were obtained by fusing mouse sple