抗人蛋白酶激活受体3单克隆抗体的制备与鉴定

发布时间：2018-03-29 19:04

本文选题：人蛋白酶激活受体3　切入点：单克隆抗体　出处：《汕头大学》2007年硕士论文

【摘要】： 背景与目的：蛋白酶激活受体3(protease-activated receptor-3，PAR3)属于G蛋白偶联受体(G protein-coupled receptor，GPCR)家族成员，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。PAR3存在于单核细胞、巨噬细胞、树突状细胞等，广泛分布在心脏，肝脏，胰腺，胸腺，小肠，胃，淋巴结，气管，肾上腺和骨髓等各种组织，参与炎症、肿瘤等疾病。制备hPAR3抗体，有利于检测PAR3的表达，，深入了解其在疾病中的作用。鉴于我国国情，研制效价高，特异性好的PAR3单克隆抗体(mAb)，具有重要意义。方法：以hPAR3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hPAR3 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、免疫组织化学染色、流式细胞分析、激光共聚焦扫描显微镜、Dot blot等技术鉴定mAb的特异性。结果：获得2株可稳定分泌抗hPAR3 mAb的杂交瘤细胞，其分泌的mAb均为IgM。免疫组化染色表明，mAb与肺中的巨噬细胞，结肠中的淋巴及疑似肥大细胞，扁桃体和包皮组织的淋巴细胞呈阳性反应。流式细胞仪分析及激光共聚焦扫描显微镜观察显示，2株mAb与人肺腺癌细胞系A549细胞上的PAR3呈阳性反应。Dot blot分析表明，其mAb能与转印至NC膜上的PAR3Ag有效结合。结论：成功地制备抗hPAR3 mAb，为研究PAR3的表达，作用及其相关工作提供了有用的试剂。
[Abstract]:Background & AIM: protease activated receptor 3(protease-activated receptor-3 (PAR3) is a member of G protein coupled receptor G protein-coupled receptor (GPCR3) family. PAR3 is composed of N- terminal and extracellular loop, transmembrane region (7 transmembrane spirals) and intracellular region (intracellular loop and C- terminal). PAR3 exists in monocytes, macrophages, dendritic cells and so on, and is widely distributed in heart, liver, pancreas, etc. Thymus, small intestine, stomach, lymph node, trachea, adrenal gland and bone marrow participate in inflammation, tumor and other diseases. The preparation of hPAR3 antibody is helpful to detect the expression of PAR3 and to understand its role in the disease. It is of great significance to develop PAR3 monoclonal antibody with high titer and good specificity. Methods: BALB/c mice were immunized with hPAR3. The hybridoma cells secreting specific anti-#en2# mAb were screened by indirect ELISA method. The IgG subclasses of mAb were identified by Ig and subclass identification kit, and the Ig subclasses of mAb were identified by immunohistochemical staining. The specificity of mAb was identified by flow cytometry, laser confocal scanning microscope (LSCM) and dot blot. Results: two hybridoma cells secreting hPAR3 mAb stably were obtained. The mAb secreted by them were all IgM.Immunohistochemical staining showed that mAb was associated with macrophages in lung, lymph nodes and suspected mast cells in colon. The positive reaction of lymphocytes in tonsils and prepuce tissues was observed by flow cytometry and confocal laser scanning microscopy. The positive reaction between mAb and PAR3 in human lung adenocarcinoma cell line A549 was observed by flow cytometry and laser confocal scanning microscopy. Its mAb can be effectively combined with PAR3Ag transferred to NC film. Conclusion: the successful preparation of anti hPAR3 mAbprovides a useful reagent for studying the expression, effect and related work of PAR3.
【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

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