蛋白酶活化受体1单克隆抗体制备及鉴定.docVIP

蛋白酶活化受体1单克隆抗体制备及鉴定 　 作者：马文静，何韶衡，周艳春，陈玉珍，黄阗，方泽漫，陈霖霏 【摘要】 目的 制备蛋白酶活化受体1(PAR1)单克隆抗体(mAb)。方法 以PAR1特异性片段为免疫原(13肽)免疫BALB/c小鼠，运用杂交瘤技术制备抗PAR1 mAb。采用捕获酶联免疫吸附试验(capture ELISA)鉴定mAb的Ig亚类；通过ELISA、Dot blot、免疫组化染色法、流式细胞仪分析、激光共聚焦显微镜技术鉴定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。免疫组化染色表明，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细胞胞浆内及细胞膜上的PAR1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在A549细胞胞浆内及细胞膜上均可见。结论 成功地制备出抗PAR1 mAb，为进一步研究炎症性疾病提供有用的试剂。 【关键词】 蛋白酶活化受体1；单克隆抗体；酶联免疫吸附试验 　　Production and identification of monoclonal antibodies　against proteinase activated receptor1 　　ABSTRACT: Objective To produce and identify antiPAR1 monoclonal antibodies. Methods BALB/c mice were immunized with 13 amino acid peptide of PAR1 and hybridoma cell lines were obtained by hybridoma technology. The specificity of mAbs was characterized by capture enzymelinked immunosorbent assay (ELISA), dot blot, immunohistochemical staining, flow cytometry (FCM) and confocal laser scanning microscopy (CLSM). Results Two hybridoma cell lines that secreted mAbs against PAR1 were obtained. One mAb was IgM, and the other was IgG2b. Immunohistochemical staining revealed that the reactivities of mAbs to the lymphocyte cells in tonsil and colon tissues, to fibroblast cells in foreskin tissues, to macrophages in lung tissues were observed. FCM showed that the 2 mAbs reacted to PAR1 expressed both on the membrane surface and in the cytoplasm of A549 cells. CLSM examination showed that the fluorescent markers were located both on the membrane and in the cytoplasm of A549 cells. Conclusion The mAbs against PAR1 are prepared successfully, which provides a valuable tool for studies on inflammatory diseases. 　　KEY WORDS: proteinase activated receptor1; monoclonal antibodies; enzyme linked immunosorbsent assay 　　蛋白酶活化受体1(proteinase activated receptor1, PAR1)是一种蛋白质，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[1]。受体由细胞外区(N末端和细胞外袢)、跨膜区(7个跨膜螺旋)及细胞内区(细胞内袢和C末端)组成 。C末端与受体脱敏和信号转导有关；N末端有相应受体的蛋白酶裂解位点。