【作者】 吴先强；

【导师】 郭泽坤； 雷鸣；

【作者基本信息】 西北农林科技大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 早幼粒细胞白血病蛋白核体(Promyelocytic leukaemia nuclear bodies,PML-NBs)是哺乳动物细胞核中的一种亚核结构,广泛参与如转录调节、基因组稳定性、抗病毒感染、抑制肿瘤、细胞凋亡、细胞衰老等许多重要的细胞生命活动。早幼粒细胞白血病蛋白PML(Promyelocytic leukaemil)是成熟PML NB的一种重要分子。SUMO(small ubiquitin-like modifier)是近年来发现的在结构上类似于泛素的一类小分子蛋白质修饰物,SUMO通过对底物赖氨酸残基的修饰,调节蛋白质-蛋白质相互作用、蛋白亚细胞定位等。PML-NB的形成依赖于SUMO对其3个赖氨酸位点的修饰,被SUMO化修饰后,PML蛋白之间发生相互作用形成聚集体,并募集多种蛋白到其上,如P53,Daxx,SP100,CBP,Mdm2,AP-1,等,这些蛋白在细胞核内发挥着重要功能。本试验以PML家族成员PML-Ⅲ为研究对象。首先通过PCR从pEGFP-PML-Ⅲ扩增PML-Ⅲ1至600bp的基因序列,然后构建了PML-Ⅲ1至600bp的原核表达载体,获得重组质粒pGEX-4T-1(+)-PML-Ⅲ( 1-600);扩增PML-Ⅲ全长基因,构建真核表达载体,获得重组质粒pCMV-HA-PML-Ⅲ、pCMV-Myc-PML-Ⅲ。将pGEX-4T-1(+)-PML-Ⅲ( 1-600)转化大肠杆菌表达菌株BL21(DE3)plysS,IPTG诱导表达,SDS-PAGE和Western Blot结果表明融合蛋白GST-PML(1~200AA)在BL21 (DE3) plysS中得到了高效表达,融合蛋白表观分子量约为46kDa。经GST亲和柱层析进一步纯化获得了可溶性重组蛋白。以纯化的融合蛋白GST-PMLⅢ(1~200AA)作为抗原免疫家兔制备多克隆抗体,并利用ELISA、Western blot检测抗体的灵敏度和特异性。结果说明原核载体构建成功,ELISA结果为阳性,多克隆抗体效价为1:128000;可以特异性识别内源性PMLⅢ蛋白。将真核表达载体pEGFP-C1-PML-Ⅲ利用脂质体转染法转染到Hela细胞中瞬时表达,在荧光显微镜下观察PML-Ⅲ蛋白的亚细胞结构定位。将真核表达载体pDsRed-C1-SUMO-1和pEGFP-C1-PML-Ⅲ利用脂质体转染法共转染到Hela细胞中进行瞬时表达,在荧光显微镜下观察蛋白的共定位情况。结果说明真核载体在Hela细胞内成功表达;PML-Ⅲ蛋白与SUMO-1蛋白共定位在核内。更多还原

【Abstract】 Promyelocytic leukaemia nuclear bodies(PML-NBs)are subnuclear structures of mammalian cell nucleus,involved in the regulation of diverse cellular functions such as transciptional regulation,genomic stability,anti-viral infection,tumour suppression,apoptosis,cellular senescence. Promyelocytic leukaemia(PML) protein is a crucial molecule of mature PML-NB. SUMO are recently discovered small ubiquitin-like modiers. SUMO modulate the protein-protein interaction,protein localization by covalently modify to the lysine residue of target protein. Sumoylation of the three lysine residue of PML is necessary for the formation of PML-NB. fter modification by SUMO,PML protein interacts and assembles together,recruiting many proteins such as P53,Daxx,SP100,CBP,Mdm2 and so on. These protein play many important functions in cell nucleus.In this study,we choose PMLⅢ,one member of PML family as subject. First,we cloned N terminal 1 to 600bp sequence of PMLⅢby PCR from pEGFP-PMLⅢ. The amplicating sequences of PMLⅢwere inserted into vectors to obtain prokaryotic expression vector pGEX-4T-1-PML(1~200AA) and eukaryotic expression vectors pCMV-HA-PMLⅢ,pCMV-Myc-PMLⅢ,respectively.The recombinant vector pGEX-4T-1-PML(1~200AA) was transformed into E. coli BL21 (DE3) plysS. The fusion protein about 46kDa was detected by SDS-PAGE and Western blot in the induced recombinant BL21 (DE3) plysS strain. The fusion protein GST-PML(1~200AA) was purified by the GST affinity chromatography column. Rabbits were immunized with the purified protein,and the antiserum was obtained,processed by ELISA,Western-blot to detect the sensitivity and specificity of its antibody. Result indicated that,prokaryotic expression vector pGEX-4T-1-PML(1~200AA) was successful. The result of ELISA was positive,and the valence of antibody was 1:128000. Western blot confirmed that the antiserum reacted specifically to the PML protein.Eukaryotic expression vector pEGFP-C1-PML-Ⅲand pDsRed-C1-SUMO-1 were co-transfected into Hela cell by liposome and expressed transiently,then we observed sub-cellular co-localization of protein PMLⅢand SUMO-1 by fluorescence microscope. Eukaryotic expression vectors pCMV-HA-PML-Ⅲ,pCMV-Myc-PML-Ⅲwere transfected into Hela cell by liposome and expressed transiently,then we use Western-blot to detect the expression. The vectors pCMV-HA-PMLⅢ,pCMV-Myc-PMLⅢwere expressed in Hela successfully.更多还原