抗蛋白酶激活受体-4单克隆抗体的制备与鉴定
发布时间：2018-11-20 05:35
【摘要】： 背景与目的:Kahn和Xu于1998年成功克隆了PAR4基因[1],并从淋巴瘤Daudi细胞中得到了它的序列,全长为4.9kb。其由385个氨基酸组成,和其它3种PARs有33%的同源性,细胞外包含一个推定的Arg/Gly丝氨酸蛋白酶切割位点, Northern blot显示PAR4的mRNA表达于人类的很多组织:肺脏上高表达,胰脏,甲状腺,睾丸及小肠亦表达;荧光原位杂交实验显示人的PAR4基因位于19p12染色体。但是PAR4在炎症及其他疾病中的确切作用及其机制还需要进一步的研究,因此我们制备廉价、有效的抗PAR-4 mAb具有重要意义。 方法:以受Fmoc树脂保护PAR-4特异性合成多肽(13肽)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PAR-4 mAb的杂交瘤细胞;采用capture ELISA法鉴定mAb的Ig亚类;通过ELISA、Dot 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:结果:获得3株可稳定分泌抗PAR4 mAb的杂交瘤细胞,其分泌的mAb均为IgM;免疫组化染色表明,mAb将人结肠组织中的腺上皮细胞、疑似肥大细胞和淋巴细胞,扁桃体中的淋巴细胞和包皮组织中的肥大细胞染成褐色;流式细胞术分析显示,3株mAb与A549细胞的PAR4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细胞的胞浆和胞膜上。 结论:成功地制备抗PAR4 mAb,为血液疾病、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: PAR4 gene was cloned successfully by Kahn and Xu in 1998 and its sequence was obtained from lymphoma Daudi cells with a total length of 4.9 kb. It is composed of 385 amino acids and has 33% homology with three other PARs species. The extracellular, Northern blot containing a presumed Arg/Gly serine protease cleavage site shows that PAR4 mRNA is expressed in many human tissues: high expression in lung, high expression in pancreas, Thyroid, testis and small intestine were also expressed. Fluorescence in situ hybridization showed that the human PAR4 gene was located on the 19p12 chromosome. However, the exact role of PAR4 in inflammation and other diseases and its mechanism need further study. Therefore, it is of great significance to prepare cheap and effective anti-PAR-4 mAb. Methods: BALB/c mice were immunized with PAR-4 specific synthetic polypeptide (13 peptide) protected by Fmoc resin. Spleen cells and myeloma NS-1 cells were fused. The hybridoma cells secreting specific anti-PAR-4 mAb were screened by indirect ELISA method, and the Ig subclasses of mAb were identified by capture ELISA method. The specificity of mAb was identified by ELISA,Dot blot, immunohistochemical staining, flow cytometry and confocal laser scanning microscopy. Results: three hybridoma cells stably secreting anti PAR4 mAb were obtained, and the mAb secreted by them were all IgM;. Immunohistochemical staining showed that the glandular epithelial cells, suspected mast cells and lymphocytes in human colon, lymphocytes in tonsils and mast cells in prepuce tissue were stained brown by mAb. The results of flow cytometry showed that the PAR4 of the three mAb and A549 cells were positive, and the fluorescence labeled positive reaction was mainly located on the cytoplasm and membrane of A549 cells by confocal laser scanning microscopy. Conclusion: the successful preparation of anti-PAR4 mAb, provides useful reagents for the research of blood diseases, allergic diseases 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】：2008
【分类号】：R392
本文编号：2343927
[Abstract]:Background & AIM: PAR4 gene was cloned successfully by Kahn and Xu in 1998 and its sequence was obtained from lymphoma Daudi cells with a total length of 4.9 kb. It is composed of 385 amino acids and has 33% homology with three other PARs species. The extracellular, Northern blot containing a presumed Arg/Gly serine protease cleavage site shows that PAR4 mRNA is expressed in many human tissues: high expression in lung, high expression in pancreas, Thyroid, testis and small intestine were also expressed. Fluorescence in situ hybridization showed that the human PAR4 gene was located on the 19p12 chromosome. However, the exact role of PAR4 in inflammation and other diseases and its mechanism need further study. Therefore, it is of great significance to prepare cheap and effective anti-PAR-4 mAb. Methods: BALB/c mice were immunized with PAR-4 specific synthetic polypeptide (13 peptide) protected by Fmoc resin. Spleen cells and myeloma NS-1 cells were fused. The hybridoma cells secreting specific anti-PAR-4 mAb were screened by indirect ELISA method, and the Ig subclasses of mAb were identified by capture ELISA method. The specificity of mAb was identified by ELISA,Dot blot, immunohistochemical staining, flow cytometry and confocal laser scanning microscopy. Results: three hybridoma cells stably secreting anti PAR4 mAb were obtained, and the mAb secreted by them were all IgM;. Immunohistochemical staining showed that the glandular epithelial cells, suspected mast cells and lymphocytes in human colon, lymphocytes in tonsils and mast cells in prepuce tissue were stained brown by mAb. The results of flow cytometry showed that the PAR4 of the three mAb and A549 cells were positive, and the fluorescence labeled positive reaction was mainly located on the cytoplasm and membrane of A549 cells by confocal laser scanning microscopy. Conclusion: the successful preparation of anti-PAR4 mAb, provides useful reagents for the research of blood diseases, allergic diseases 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】：2008
【分类号】：R392
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