發(fā)布時間：2018-06-27 06:02

  本文選題：PML + PML(NLS-)　； 參考：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：第一部分NLS在PMI,定位及其與importin a相互作用機(jī)制研究目的通過間接免疫熒光技術(shù)驗證PNL、PML(NLS-)胞內(nèi)定位,以及通過間接免疫熒光技術(shù)和免疫共沉淀技術(shù)驗證PML/PML(NLS-)與importin α之間的相互作用。方法構(gòu)建真核表達(dá)質(zhì)粒pCMV-HA-PML、pCMV-HA-PML (NLS-) 和pCMV-Myc-importin α并共轉(zhuǎn)染HEK 293細(xì)胞,利用間接免疫熒光檢測PML、PML(NLS-)胞內(nèi)定位；利用免疫熒光雙標(biāo)法和免疫共沉淀分別驗證PML/PML(NLS-)與importin α之間的相互作用；同時提取原代APL細(xì)胞、non-APL病人及正常人的中性粒細(xì)胞,運用Western blot,免疫熒光,檢測中性粒細(xì)胞內(nèi)PML(NLS-)蛋白的表達(dá)與定位。結(jié)果成功構(gòu)建真核質(zhì)粒pCMV-HA-PML、pCMV-HA-PML (NLS-)和pCMV-Myc-importin α;免疫熒光顯示PML定位與胞核中呈斑點狀,PML(NLS-)主要定位子胞漿；免疫共沉淀顯示,使用兔抗HA多克隆抗體沉淀與HA-PML相互作用的蛋白,再用鼠抗Myc單克隆抗體進(jìn)行Western blott檢測,可以檢測mportin α蛋白；沉淀HA-PML(NLS-)相互作用的蛋白則不可以檢測到importin α蛋白；激光共聚焦顯微鏡下可觀察到PML與importin α共定位于胞核內(nèi),PML(NLS-)與importin α不存在共定位；原代APL細(xì)胞表達(dá)PML (NLS-)蛋白并定位于細(xì)胞胞漿。而非APL病人和正常人的中性粒細(xì)胞胞漿中幾乎沒有PML (NLS-)表達(dá),僅表達(dá)PML蛋白并定位于胞核。結(jié)論：PML蛋白通過其NLS序列與importin α相互作用介導(dǎo)入核；PML (NLS-)可以作為APL診斷標(biāo)志。第二部分抗PML蛋白核定位信號(NLS)抗體的制備及其在APL診斷中的應(yīng)用研究目的：以NLS多肽作為免疫原,制備抗PML蛋白抗體。方法：分析PML蛋白NLS序列免疫原性,合成NLS多肽；以此多肽作為免疫原,免疫三只小鼠,兩周后加強(qiáng)免疫：末次免疫10天后取尾血制備抗體血清,以未免疫小鼠血清為對照,ELISA檢測抗體效價；WESTERN BLOT與間接免疫熒光驗證抗體特異性。結(jié)果：三支抗體血清ELISA檢測均有陽性反應(yīng),其中2號抗體血清效價最高達(dá)1：50000,以2號抗體血清為一抗,western blot檢測野生型PML蛋白分子量大小約為70KDa；免疫熒光顯示PML位于胞核中呈斑點狀。結(jié)論：成功制備了抗PML蛋白核定位信號序列的抗體,區(qū)別檢測野生型PML蛋白和PML(NLS-)蛋白為APL臨床診斷提供新的依據(jù)。
[Abstract]:The first part is about the localization of PML and its interaction with importin a. Objective to verify the intracellular localization of PML (NLS-) by indirect immunofluorescence technique, and to verify the interaction between PML / PML (NLS-) and importin 偽 by indirect immunofluorescence and co-immunoprecipitation. Methods Eukaryotic expression plasmids pCMV-HA-PML (NLS-) and pCMV-Myc-importin 偽 were constructed and cotransfected into HEK293 cells. The expression and localization of PML (NLS-) protein in primary APL cells from non-APL patients and normal controls were detected by Western blotand immunofluorescence. Results Eukaryotic plasmids pCMV-HA-PML (NLS-) and pCMV-Myc-importin 偽 were successfully constructed, and immunofluorescence showed that PML was mainly located in the cytoplasm of HA-PML, and immunoprecipitation showed that rabbit anti-HA polyclonal antibody was used to precipitate proteins interacting with HA-PML. The mportin 偽 protein could be detected by Western blott with mouse anti-Myc monoclonal antibody, but importin 偽 protein could not be detected by precipitating HA-PML (NLS-) interaction protein. Under laser confocal microscopy, it was observed that PML and importin 偽 were not co-located in the nucleus and the primary APL cells expressed PML- (NLS-) protein and located in the cytoplasm. However, there was almost no PML (NLS-) expression in the cytoplasm of neutrophils from non-APL patients and normal controls. Only PML protein was expressed and localized in the nucleus. Conclusion the importin 偽 protein can be used as a diagnostic marker of importin. The second part: preparation of anti-PML protein nuclear localization signal (NLS) antibody and its application in the diagnosis of APL objective: to prepare anti-PML protein antibody using NLS peptide as immunogen. Methods: the immunogenicity of PML protein NLS sequence was analyzed and NLS peptide was synthesized, which was used as immunogen to immunize three mice. The antibody titers of unimmunized mice were detected by Elisa, and the specificity of antibodies was verified by indirect immunofluorescence. Results: all the three antibodies were detected by Elisa. The highest titer of antibody 2 was 1: 500, and the molecular weight of wild-type blot was about 70KDa. Immunofluorescence showed that PML was spotted in the nucleus. Conclusion: the antibody against PML nuclear localization signal sequence was successfully prepared. The detection of wild type PML protein and PML (NLS-) protein provides a new basis for clinical diagnosis of APL.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R733.71

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王建榮,楊林;腎病理免疫熒光組織變性的預(yù)防[J];現(xiàn)代中西醫(yī)結(jié)合雜志;2004年12期

2 王建榮;免疫熒光組織變性的原因及對策[J];中國誤診學(xué)雜志;2004年08期

3 朱厚礎(chǔ);朱德鐘;;免疫熒光自動化的一些進(jìn)展[J];國外醫(yī)學(xué).臨床生物化學(xué)與檢驗學(xué)分冊;1981年01期

4 謝正e，

本文編號：2072922