PML-RARα中NLS在急性早幼粒细胞白血病临床诊断中的实验研究

发布时间：2018-06-27 06:02

本文选题：PML + PML(NLS-)　；参考：《重庆医科大学》2015年硕士论文

【摘要】：第一部分NLS在PMI,定位及其与importin a相互作用机制研究目的通过间接免疫荧光技术验证PNL、PML(NLS-)胞内定位,以及通过间接免疫荧光技术和免疫共沉淀技术验证PML/PML(NLS-)与importin α之间的相互作用。方法构建真核表达质粒pCMV-HA-PML、pCMV-HA-PML (NLS-) 和pCMV-Myc-importin α并共转染HEK 293细胞,利用间接免疫荧光检测PML、PML(NLS-)胞内定位；利用免疫荧光双标法和免疫共沉淀分别验证PML/PML(NLS-)与importin α之间的相互作用；同时提取原代APL细胞、non-APL病人及正常人的中性粒细胞,运用Western blot,免疫荧光,检测中性粒细胞内PML(NLS-)蛋白的表达与定位。结果成功构建真核质粒pCMV-HA-PML、pCMV-HA-PML (NLS-)和pCMV-Myc-importin α;免疫荧光显示PML定位与胞核中呈斑点状,PML(NLS-)主要定位子胞浆；免疫共沉淀显示,使用兔抗HA多克隆抗体沉淀与HA-PML相互作用的蛋白,再用鼠抗Myc单克隆抗体进行Western blott检测,可以检测mportin α蛋白；沉淀HA-PML(NLS-)相互作用的蛋白则不可以检测到importin α蛋白；激光共聚焦显微镜下可观察到PML与importin α共定位于胞核内,PML(NLS-)与importin α不存在共定位；原代APL细胞表达PML (NLS-)蛋白并定位于细胞胞浆。而非APL病人和正常人的中性粒细胞胞浆中几乎没有PML (NLS-)表达,仅表达PML蛋白并定位于胞核。结论：PML蛋白通过其NLS序列与importin α相互作用介导入核；PML (NLS-)可以作为APL诊断标志。第二部分抗PML蛋白核定位信号(NLS)抗体的制备及其在APL诊断中的应用研究目的：以NLS多肽作为免疫原,制备抗PML蛋白抗体。方法：分析PML蛋白NLS序列免疫原性,合成NLS多肽；以此多肽作为免疫原,免疫三只小鼠,两周后加强免疫：末次免疫10天后取尾血制备抗体血清,以未免疫小鼠血清为对照,ELISA检测抗体效价；WESTERN BLOT与间接免疫荧光验证抗体特异性。结果：三支抗体血清ELISA检测均有阳性反应,其中2号抗体血清效价最高达1：50000,以2号抗体血清为一抗,western blot检测野生型PML蛋白分子量大小约为70KDa；免疫荧光显示PML位于胞核中呈斑点状。结论：成功制备了抗PML蛋白核定位信号序列的抗体,区别检测野生型PML蛋白和PML(NLS-)蛋白为APL临床诊断提供新的依据。
[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 伪 by indirect immunofluorescence and co-immunoprecipitation. Methods Eukaryotic expression plasmids pCMV-HA-PML (NLS-) and pCMV-Myc-importin 伪 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 伪 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 伪 protein could be detected by Western blott with mouse anti-Myc monoclonal antibody, but importin 伪 protein could not be detected by precipitating HA-PML (NLS-) interaction protein. Under laser confocal microscopy, it was observed that PML and importin 伪 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 伪 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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.71

【相似文献】