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| 目的：通过研究蛋白质相互作用的方法，验证MyD88与Siah相互作用的真实性，并对其相互作用的意义做初步的探讨。方法：本实验室通过酵母双杂交技术先初步筛选到了472对相互作用对。后来，采用无酶克隆大规模构建了荧光共定位载体。共转染哺乳动物细胞Hela（或293T），筛选到了一系列相互作用对。由于MyD88是TLR信号通路中的重要接头分子，关于他的研究意义很大，同时Siah的生化性质比较明确，所以我们挑选了MyD88与Siah1做进一步的研究。做了免疫共沉淀，双荧光报告基因检测及体内泛素化检测等实验。结果：在外转质粒的情况下，免疫荧光共定位实验结果为阳性，并鉴定了MyD88与Siah互作的结构域是DBD域。在293T细胞中，外转质粒时，免疫共沉淀的结果也为阳性。双荧光报告基因检测初步显示Siah对TLR信号通路的影响为负调控。 |
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目的：通过研究蛋白质相互作用的方法，验证MyD88与Siah相互作用的真实性，并对其相互作用的意义做初步的探讨。方法：本实验室通过酵母双杂交技术先初步筛选到了472对相互作用对。后来，采用无酶克隆大规模构建了荧光共定位载体。共转染哺乳动物细胞Hela（或293T），筛选到了一系列相互作用对。由于MyD88是TLR信号通路中的重要接头分子，关于他的研究意义很大，同时Siah的生化性质比较明确，所以我们挑选了MyD88与Siah1做进一步的研究。 Objective: According the methods for research on protein-protein interaction, the authenticity of interaction between MyD88 and Siah was verified, and the significance of the interaction was discussed preliminary as well. Methods: the 472 pairs of interaction partners were screened by means of yeast-two hybrid technique in our laboratory. Subsequently, the fluorescent co-localization vector was constructed in large by means of enzyme-free cloning. A series of interaction partners were screened from transfection of Mammalian cells Hela (or 293T). Because MyD88 was an important adaptor molecule in TLR signaling pathways, so on which the research showed great significance, while the biochemical nature of Siah was very clear, so MyD88 and Siah1 were further studied. |
2楼2009-05-31 09:38:14
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做了免疫共沉淀，双荧光报告基因检测及体内泛素化检测等实验。结果：在外转质粒的情况下，免疫荧光共定位实验结果为阳性，并鉴定了MyD88与Siah互作的结构域是DBD域。在293T细胞中，外转质粒时，免疫共沉淀的结果也为阳性。双荧光报告基因检测初步显示Siah对TLR信号通路的影响为负调控。 Immunoprecipitation, dual fluorescent report gene detection and in vivo ubiquitination was tested. Results: In the case of plasmid outer transfer, immunofluorescence co-localization results was positive, and domain from interaction between MyD88 and Siah was identified as DBD domain. In 293T cells, outside the transfer plasmid, the results of immunoprecipitation was positive as well. Dual-fluorescence report gene detection initially indicated that effect of Siah on TLR signaling pathway exhibited negative regulation. |
3楼2009-05-31 10:03:32
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水过无恒(金币+50,VIP+0):多谢了，不过感觉还是不够“地道”，金币奉上，请笑纳 5-31 12:13
水过无恒(金币+50,VIP+0):多谢了，不过感觉还是不够“地道”，金币奉上，请笑纳 5-31 12:13
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汇总起来，仅供参考 Objective: According the methods for research on protein-protein interaction, the authenticity of interaction between MyD88 and Siah was verified, and the significance of the interaction was discussed preliminary as well. Methods: the 472 pairs of interaction partners were screened by means of yeast-two hybrid technique in our laboratory. Subsequently, the fluorescent co-localization vector was constructed in large by means of enzyme-free cloning. A series of interaction partners were screened from transfection of Mammalian cells Hela (or 293T). Because MyD88 was an important adaptor molecule in TLR signaling pathways, so on which the research showed great significance, while the biochemical nature of Siah was very clear, so MyD88 and Siah1 were further studied. Immunoprecipitation, dual fluorescent report gene detection and in vivo ubiquitination was tested. Results: In the case of plasmid outer transfer, immunofluorescence co-localization results was positive, and domain from interaction between MyD88 and Siah was identified as DBD domain. In 293T cells, outside the transfer plasmid, the results of immunoprecipitation was positive as well. Dual-fluorescence report gene detection initially indicated that effect of Siah on TLR signaling pathway exhibited negative regulation. |
4楼2009-05-31 10:10:49