NE介导的PML-RARα融合蛋白裂解产物在APL发生中的分子机制研究
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摘要
第一部分NLS-RARα与JTV1蛋白的细胞内共定位实验
目的:利用间接免疫荧光技术和激光共聚焦显微镜观察NLS-RARα和JTV1蛋白在哺乳动物细胞内是否共定位表达。
方法:构建真核细胞表达载体pCMV-HA-NLS-RARα和pCMV-Myc-JTV1,经酶切鉴定后,共转染人胚肾293细胞,利用间接免疫荧光技术研究它们的表达是否存在细胞内共定位。
结果:构建的重组表达载体pCMV-HA-NLS-RARα和pCMV-Myc-JTV1经酶切鉴定成功,转染HEK293细胞。HA-NLS-RARα蛋白用抗HA的一抗和Cy3结合的羊抗兔IgG标记,Myc-JTV1蛋白用抗Myc的一抗和FITC结合的羊抗鼠IgG标记,激光共聚焦显微镜下可观察到两种蛋白的共定位表达。
结论:成功构建真核表达载体pCMV-HA-NLS-RARα和pCMV-Myc-JTV1,在HEK293细胞表达后利用间接免疫荧光技术证实NLS-RARα和JTV1蛋白在核内可共定位表达,从而进一步提示二者在细胞核内存在相互作用的可能。
第二部分早幼粒细胞白血病基因PML变异蛋白全长及其结构域诱饵载体的构建及鉴定
目的:为研究中性粒细胞弹性蛋白酶(NE)酶切PML-RARα融合蛋白后产生的早幼粒细胞白血病基因(PML)的变异蛋白和其中含环指/B-BOX结构与含coiled-coil结构的两个结构域的功能,构建含其变异蛋白全长及结构域序列的诱饵表达载体,为进一步应用酵母双杂交技术筛选与之相互作用的蛋白建立实验基础。
方法:PCR扩增mut-PML全长及其两个结构域序列,克隆入诱饵载体pGBKT7中,经测序鉴定后,将诱饵载体转化到酵母细胞AH109中,检测诱饵蛋白有无毒性,渗漏和自激活作用,同时利用蛋白印迹法分析诱饵蛋白的表达。
结果:成功扩增mut-PML全长及其两个结构域的基因片段,并正确克隆入pGBKT7中。诱饵载体成功转化到酵母细胞AH109中,其中诱饵蛋白BD-mut-PML , BD-PML-B无毒性,但具有渗漏和自激活作用,诱饵蛋白BD-PML-C无毒性,渗漏和自激活作用,蛋白印迹法分析证实酵母细胞表达诱饵蛋白。
结论:mut-PML全长和含环指/B-BOX的结构域具有转录因子活性;成功构建含coiled-coil结构的PML结构域的酵母诱饵表达载体。
第三部分酵母双杂交技术筛选在胞内与PML-C结构域相互作用的蛋白
目的:利用酵母双杂交技术筛选与PML-C结构域相互作用的蛋白质,为研究野生型PML和其变异体可能的作用靶点及其分子作用机制奠定基础。
方法:通过诱饵质粒pGBKT7-PML-C,利用酵母双杂交系统从白血病细胞cDNA文库中筛选与PML-C结构域相互作用的蛋白质。
结果:利用酵母双杂交技术初步筛选到43个能与PML-C结构域相互作用的克隆;经进一步的归类与酵母回转试验得到9个阳性克隆。
结论:在细胞内PML-C结构域能与多种蛋白质有相互作用。NE介导的APL的发生可能与这些相互作用所致的生物学功能改变有关。
PARTⅠTHE STUDY ON COLOCALIZATION OF NLS-RARαAND JTV1 INTRACELLULAR LOCALIZATION IN CELLS
Objective: To explore colocalization of NLS-RARαand JTV1 in mammalian cell through indirect immunofluorescence technique and laser- confocal microscopy.
Methods: HA-tagged fusion protein (pCMV-HA-NLS-RARα) expression vector and Myc-tagged fusion protein (pCMV-Myc-JTV1) expression vector were respectively constructed, identified and transfected into human embryo kidney 293(HEK293) cells. The colocalization of NLS-RARαand JTV1 was observed by indirect immunofluorescence technique.
Results: Double restriction enzyme digestion show that pCMV-HA-NLS-RARαand pCMV-Myc-JTV1 were successfully constructed. When HA-NLS-RARαwas tagging by anti-HA polyclonal antibody and Cy3-conjugated goat anti-rabbit IgG,Myc-JTV1 was marking by anti-Myc monoclonal antibody and FITC-conjugated goat anti-mouse IgG, the intracellular colocalization of NLS-RARαand JTV1 was detected by indirect immunofluorescence technique.
Conclusion: The bait expression vector of was constructed successfully. HA-NLS-RARαprotein and Myc-JTV1 protein, their intracellular localization in nucleus was analyzed using indirect immunofluorescence, is implying that there is possible interaction between the two proteins in nucleus.
PARTⅡConstruction, expression and identification of bait vector of MUT-PML and the two structural domain of MUT-PML
Objective: To investigate the functions of mut-PML and the two structural domains of mut-PML, one domain with ring finger/B-BOX structure and the other one with coiled-coil structure, construct bait expression vector of mut-PML and the two domains for screening the target proteins interacting with the bait protein through the yeast two-hybrid system.
Methods: The fragments of mut-PML and the two structural domain of mut-PML were amplified by PCR, and then were cloned into the bait expression vector pGBKT7. After being verified by sequencing, the bait vectors were transformed into AH109 yeast strain. Toxicity, leakage and self-activation of the bait proteins were detected. The expression of the bait protein was analyzed by Western blot.
Results: The fragments of mut-PML and the two structural domain of mut-PML were amplified and cloned into pGBKT7 successfully. The bait vectors were transformed into AH109, the bait proteins,BD-mut-PML and BD-PML-B have no toxicity but leakage and self-activation were found, the other one, BD-PML-C has no toxicity, leakage and self-activation. The expression of the bait protein was confirmed by Western blot.
Conclusion: The domain of mut-PML with ring finger/B-BOX structure has activity of transcription, the transcription activity of full-length mut-PML is due to the domain. The domain of mut-PML with coiled-coil structure was constructed successfully, which layed the foundations for screening target proteins interacting with the bait protein using the yeast two-hybrid technique.
PARTШSCREENING TARGET PROTEINS INTERACTING WITH PML-C BY YEAST TWO-HYBRID SYSTEM
Objective: To screen the protein interacting with the domain of PML with coiled-coil structure (PML-C) via the yeast two-hybrid technique, it helps to find out the target proteins interacted with PML-C and further to study the biological function and mechanism of action.
Methods: The bait vector of pGBKT7- PML-C was constructed for screening the proteins interacting with PML-C in the leukemic cell cDNA expression library via yeast two-hybrid technique.
Results: 43 proteins were screened interacting with PML-C by yeast two-hybrid technique, nine positive clones were identified by retransformation in yeast.
Conclusion: There are some proteins interacting with PML-C in cell. The pathogenesis mediated by NE of leukemia maybe related to the biological function altered by the certain protein-protein interaction.
目的:利用间接免疫荧光技术和激光共聚焦显微镜观察NLS-RARα和JTV1蛋白在哺乳动物细胞内是否共定位表达。
方法:构建真核细胞表达载体pCMV-HA-NLS-RARα和pCMV-Myc-JTV1,经酶切鉴定后,共转染人胚肾293细胞,利用间接免疫荧光技术研究它们的表达是否存在细胞内共定位。
结果:构建的重组表达载体pCMV-HA-NLS-RARα和pCMV-Myc-JTV1经酶切鉴定成功,转染HEK293细胞。HA-NLS-RARα蛋白用抗HA的一抗和Cy3结合的羊抗兔IgG标记,Myc-JTV1蛋白用抗Myc的一抗和FITC结合的羊抗鼠IgG标记,激光共聚焦显微镜下可观察到两种蛋白的共定位表达。
结论:成功构建真核表达载体pCMV-HA-NLS-RARα和pCMV-Myc-JTV1,在HEK293细胞表达后利用间接免疫荧光技术证实NLS-RARα和JTV1蛋白在核内可共定位表达,从而进一步提示二者在细胞核内存在相互作用的可能。
第二部分早幼粒细胞白血病基因PML变异蛋白全长及其结构域诱饵载体的构建及鉴定
目的:为研究中性粒细胞弹性蛋白酶(NE)酶切PML-RARα融合蛋白后产生的早幼粒细胞白血病基因(PML)的变异蛋白和其中含环指/B-BOX结构与含coiled-coil结构的两个结构域的功能,构建含其变异蛋白全长及结构域序列的诱饵表达载体,为进一步应用酵母双杂交技术筛选与之相互作用的蛋白建立实验基础。
方法:PCR扩增mut-PML全长及其两个结构域序列,克隆入诱饵载体pGBKT7中,经测序鉴定后,将诱饵载体转化到酵母细胞AH109中,检测诱饵蛋白有无毒性,渗漏和自激活作用,同时利用蛋白印迹法分析诱饵蛋白的表达。
结果:成功扩增mut-PML全长及其两个结构域的基因片段,并正确克隆入pGBKT7中。诱饵载体成功转化到酵母细胞AH109中,其中诱饵蛋白BD-mut-PML , BD-PML-B无毒性,但具有渗漏和自激活作用,诱饵蛋白BD-PML-C无毒性,渗漏和自激活作用,蛋白印迹法分析证实酵母细胞表达诱饵蛋白。
结论:mut-PML全长和含环指/B-BOX的结构域具有转录因子活性;成功构建含coiled-coil结构的PML结构域的酵母诱饵表达载体。
第三部分酵母双杂交技术筛选在胞内与PML-C结构域相互作用的蛋白
目的:利用酵母双杂交技术筛选与PML-C结构域相互作用的蛋白质,为研究野生型PML和其变异体可能的作用靶点及其分子作用机制奠定基础。
方法:通过诱饵质粒pGBKT7-PML-C,利用酵母双杂交系统从白血病细胞cDNA文库中筛选与PML-C结构域相互作用的蛋白质。
结果:利用酵母双杂交技术初步筛选到43个能与PML-C结构域相互作用的克隆;经进一步的归类与酵母回转试验得到9个阳性克隆。
结论:在细胞内PML-C结构域能与多种蛋白质有相互作用。NE介导的APL的发生可能与这些相互作用所致的生物学功能改变有关。
PARTⅠTHE STUDY ON COLOCALIZATION OF NLS-RARαAND JTV1 INTRACELLULAR LOCALIZATION IN CELLS
Objective: To explore colocalization of NLS-RARαand JTV1 in mammalian cell through indirect immunofluorescence technique and laser- confocal microscopy.
Methods: HA-tagged fusion protein (pCMV-HA-NLS-RARα) expression vector and Myc-tagged fusion protein (pCMV-Myc-JTV1) expression vector were respectively constructed, identified and transfected into human embryo kidney 293(HEK293) cells. The colocalization of NLS-RARαand JTV1 was observed by indirect immunofluorescence technique.
Results: Double restriction enzyme digestion show that pCMV-HA-NLS-RARαand pCMV-Myc-JTV1 were successfully constructed. When HA-NLS-RARαwas tagging by anti-HA polyclonal antibody and Cy3-conjugated goat anti-rabbit IgG,Myc-JTV1 was marking by anti-Myc monoclonal antibody and FITC-conjugated goat anti-mouse IgG, the intracellular colocalization of NLS-RARαand JTV1 was detected by indirect immunofluorescence technique.
Conclusion: The bait expression vector of was constructed successfully. HA-NLS-RARαprotein and Myc-JTV1 protein, their intracellular localization in nucleus was analyzed using indirect immunofluorescence, is implying that there is possible interaction between the two proteins in nucleus.
PARTⅡConstruction, expression and identification of bait vector of MUT-PML and the two structural domain of MUT-PML
Objective: To investigate the functions of mut-PML and the two structural domains of mut-PML, one domain with ring finger/B-BOX structure and the other one with coiled-coil structure, construct bait expression vector of mut-PML and the two domains for screening the target proteins interacting with the bait protein through the yeast two-hybrid system.
Methods: The fragments of mut-PML and the two structural domain of mut-PML were amplified by PCR, and then were cloned into the bait expression vector pGBKT7. After being verified by sequencing, the bait vectors were transformed into AH109 yeast strain. Toxicity, leakage and self-activation of the bait proteins were detected. The expression of the bait protein was analyzed by Western blot.
Results: The fragments of mut-PML and the two structural domain of mut-PML were amplified and cloned into pGBKT7 successfully. The bait vectors were transformed into AH109, the bait proteins,BD-mut-PML and BD-PML-B have no toxicity but leakage and self-activation were found, the other one, BD-PML-C has no toxicity, leakage and self-activation. The expression of the bait protein was confirmed by Western blot.
Conclusion: The domain of mut-PML with ring finger/B-BOX structure has activity of transcription, the transcription activity of full-length mut-PML is due to the domain. The domain of mut-PML with coiled-coil structure was constructed successfully, which layed the foundations for screening target proteins interacting with the bait protein using the yeast two-hybrid technique.
PARTШSCREENING TARGET PROTEINS INTERACTING WITH PML-C BY YEAST TWO-HYBRID SYSTEM
Objective: To screen the protein interacting with the domain of PML with coiled-coil structure (PML-C) via the yeast two-hybrid technique, it helps to find out the target proteins interacted with PML-C and further to study the biological function and mechanism of action.
Methods: The bait vector of pGBKT7- PML-C was constructed for screening the proteins interacting with PML-C in the leukemic cell cDNA expression library via yeast two-hybrid technique.
Results: 43 proteins were screened interacting with PML-C by yeast two-hybrid technique, nine positive clones were identified by retransformation in yeast.
Conclusion: There are some proteins interacting with PML-C in cell. The pathogenesis mediated by NE of leukemia maybe related to the biological function altered by the certain protein-protein interaction.
引文
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