抗蛋白酶激活受体-2单克隆抗体的制备与鉴定

Preparation and characterization of monoclonal antibody against protease activated receptor-2

目的:制备出一种具有多种用途的抗蛋白酶激活受体-2(PAR-2)单克隆抗体(mAb)。方法:用人工合成的11肽PAR-2作为免疫原,采用皮下多点注射方法免疫BALB/c小鼠,取其脾细胞与NS-1骨髓瘤细胞融合,杂交瘤细胞采用间接ELISA法筛选。通过ELISA、Dot blot、免疫组化染色、流式细胞分析、激光共聚焦显微镜技术鉴定mAb的特异性。结果:获得1株可稳定分泌抗PAR-2 mAb的杂交瘤细胞。其分泌的mAb为IgM型,ELISA法检测杂交瘤细胞培养上清效价为1:16;点杂交分析表明此抗体可特异性结合PAR-2;免疫组化染色显示该单克隆抗体与人肺组织中的肺泡上皮细胞和平滑肌细胞,结肠组织中的腺上皮细胞和疑似淋巴细胞,扁桃体中的淋巴细胞,包皮组织中的鳞状上皮细胞呈阳性反应;流式细胞仪分析显示与人肺腺癌细胞系A549细胞中的PAR-2呈阳性反应;激光共聚焦扫描显微镜观察发现荧光标记的阳性反应物位于A549细胞的胞膜和胞浆。结论:成功地制备出可用于免疫组化和点杂交的抗PAR-2单克隆抗体,为PAR-2相关性疾病的研究提供了有用的工具。

Objective：To prepare and identify the monoclonal antibody（mAb）against protease activated receptor-2（PAR-2）. Methods：BLAB/c mice were immunized with specific PAR-2 polypeptide by multiple-site hypodermic injection.The spleen cells of immunized mice were fused with NS-1 myeloma cells.Indirect ELISA was used to screen the hybridoma culture supernants.The specificity of mAb were characterized by ELISA,Dot blot,immunohistochemistry,flow cytometry and confocal laser scanning microscopy（CLSM）.Results：One hybridoma cell line against PAR-2 was successfully obtained,this mAb was IgM type and the titer of antibody in supemants was 1：16.Dot blot analysis showed that the mAb could specifically bind to PAR-2.Immunohistochemical staining revealed that the reactivity of mAb to epithelial cells and smooth muscle cells in lung,epithelial cells and lymphocyte-like cells in colon,lymphocytes in tonsil and squamous epithelial cells in skin tissue.The result of flow cytometry showed that the mAb recognized PAR-2 in A549 cells.CLSM examination confirmed that the fluorescent markers were localized in both cytomembrane and cytoplasm of A549 cells.Conclusion：The mAb against PAR-2 is a useful tool for investigate PAR-2 related disease