【摘要】： 背景與目的:Kahn和Xu于1998年成功克隆了PAR4基因[1],并從淋巴瘤Daudi細(xì)胞中得到了它的序列,全長(zhǎng)為4.9kb。其由385個(gè)氨基酸組成,和其它3種PARs有33%的同源性,細(xì)胞外包含一個(gè)推定的Arg/Gly絲氨酸蛋白酶切割位點(diǎn), Northern blot顯示PAR4的mRNA表達(dá)于人類的很多組織:肺臟上高表達(dá),胰臟,甲狀腺,睪丸及小腸亦表達(dá);熒光原位雜交實(shí)驗(yàn)顯示人的PAR4基因位于19p12染色體。但是PAR4在炎癥及其他疾病中的確切作用及其機(jī)制還需要進(jìn)一步的研究,因此我們制備廉價(jià)、有效的抗PAR-4 mAb具有重要意義。 方法:以受Fmoc樹(shù)脂保護(hù)PAR-4特異性合成多肽(13肽)為免疫原免疫BALB/c小鼠,取免疫小鼠脾細(xì)胞和小鼠骨髓瘤NS-1細(xì)胞融合。通過(guò)間接ELISA法篩選可特異性分泌抗PAR-4 mAb的雜交瘤細(xì)胞;采用capture ELISA法鑒定mAb的Ig亞類;通過(guò)ELISA、Dot blot、免疫組織化學(xué)染色法、流式細(xì)胞分析、激光共聚焦掃描顯微鏡技術(shù)鑒定mAb的特異性。 結(jié)果:結(jié)果:獲得3株可穩(wěn)定分泌抗PAR4 mAb的雜交瘤細(xì)胞,其分泌的mAb均為IgM;免疫組化染色表明,mAb將人結(jié)腸組織中的腺上皮細(xì)胞、疑似肥大細(xì)胞和淋巴細(xì)胞,扁桃體中的淋巴細(xì)胞和包皮組織中的肥大細(xì)胞染成褐色;流式細(xì)胞術(shù)分析顯示,3株mAb與A549細(xì)胞的PAR4均呈陽(yáng)性反應(yīng);激光共聚焦掃描顯微鏡觀察,熒光標(biāo)記的陽(yáng)性反應(yīng)物主要位于A549細(xì)胞的胞漿和胞膜上。 結(jié)論:成功地制備抗PAR4 mAb,為血液疾病、變態(tài)反應(yīng)性疾病和炎癥性疾病的研究工作提供了有用的試劑。
[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.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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